Extracellular matrix signalling molecules

ABSTRACT

Polynucleotides encoding mammalian ECM signalling molecules affecting the cell adhesion, migration. and proliferation activities characterizing such complex biological processes as angiogenesis. chondrogenesis. and oncogenesis, are provided. The polynucleotide compositions include DNAs and RNAs comprising part. or all. of an ECM signalling molecule coding sequence, or biological equivalents. Polypeptide compositions are also provided. The polypeptide compositions comprise mammalian ECM signalling molecules, peptide fragments, inhibitory peptides capable of interacting with receptors for ECM signalling molecules, and antibody products recognizing Cyr61. Also provided are methods for producing mammalian ECM signalling molecules. Further provided are methods for using mammalian ECM signalling molecules to screen for, and/or modulate. disorders associated with angiogenesis, chondrogenesis. and oncogenesis: ex vivo methods for using mammalian ECM signalling molecules to prepare blood products are also provided.

[0001] This application claims the benefit of the filing date of U.S.provisional patent application serial No. 60/013,958, filed Mar. 15.1996.

FIELD OF THE INVENTION

[0002] The present invention is directed to materials and methodsinvolving extracellular matrix signalling molecules—polypeptidesinvolved in cellular responses to growth factors. More particularly, theinvention is directed to Cyr61-, Fisp12-, and CTGF-relatedpolynucleotides, polypeptides. compositions thereof, methods ofpurifying these polypeptides, and methods of using these polypeptides.

BACKGROUND OF THE INVENTION

[0003] The growth of mammalian cells is tightly regulated by polypeptidegrowth factors. In the adult animal, most cells are metabolically activebut are quiescent with regard to cell division. Under certainconditions, these cells can be stimulated to reenter the cell cycle anddivide. As quiescent cells reenter the active growth and division phasesof the cell cycle, a number of specific genes. the immediate earlygenes, are rapidly activated. Reentry to the active cell cycle is bynecessity tightly regulated, since a breakdown of this control canresult in uncontrolled growth, frequently recognized as cancer.Controlled reentry of particular cells into the growth phase isessential for such biological processes as angiogenesis (e.g., bloodvessel growth and repair), chondrogenesis (e.g., skeletal developmentand prosthesis integration), oncogenesis (e.g., cancer cell metastasisand tumor neovascularization), and other growth-requiring processes.

[0004] Angiogenesis. the formation of new blood vessels from theendothelial cells of preexisting blood vessels, is a complex processwhich involves a changing profile of endothelial cell gene expression,associated with cell migration, proliferation, and differentiation.Angiogenesis begins with localized breakdown of the basement membrane ofthe parent vessel. In vivo, basement membranes (primarily composed oflaminin. collagen type IV. nidogen/entactin, and proteoglycan) supportthe endothelial cells and provide a barrier separating these cells fromthe underlying stroma. The basement membrane also affects a variety ofbiological activities including cell adhesion. migration, and growthduring development and differentiation.

[0005] Following breakdown of the basement membrane. endothelial cellsmigrate away from the parent vessel into the interstitial extracellularmatrix (ECM). at least partially due to chemoattractant gradients. Themigrating endothelial cells form a capillary sprout, which elongates.This elongation is the result of migration and proliferation of cells inthe sprout. Cells located in the leading capillary tip migrate towardthe angiogenic stimulus, but neither synthesize DNA nor divide.Meanwhile, behind these leading tip cells, other endothelial cellsundergo rapid proliferation to ensure an adequate supply of endothelialcells for formation of the new vessel. Capillary sprouts then branch attheir tips, the branches anastomose or join with one another to form alumen, the basement membrane is reconstituted, and a vascular connectionis established leading to blood flow.

[0006] Alterations in at least three endothelial cell functions occurduring angiogenesis: 1) modulations of interactions with the ECM, whichrequire alterations of cell-matrix contacts and the production ofmatrix-degrading proteolytic enzymes; 2) an initial increase andsubsequent decrease in endothelial cell migration. effecting celltranslocation towards an angiogenic stimulus; and 3) a transientincrease in cell proliferation, providing cells for the growing andelongating vessel, with a subsequent return to the quiescent cell stateonce the vessel is formed. These three functions are realized byadhesive, chemotactic, and mitogenic interactions or responses,respectively. Therefore, control of angiogenesis requires interventionin three distinct cellular activities: 1) cell adhesion, 2) cellmigration, and 3) cell proliferation. Another biological processinvolving a similar complex array of cellular activities ischondrogenesis.

[0007] Chondrogenesis is the cellular process responsible for skeletalorganization. including the development of bone and cartilage.Chondrogenesis, like angiogenesis, involves the controlled reentry ofquiescent cells into the growth phase of the cell cycle. The growthphase transition is associated with altered cell adhesioncharacteristics, changed patterns of cell migration. and transientlyincreased cell proliferation. Chondrogenesis involves the initialdevelopment of chondrogenic capacity (i.e., the proto-differentiatedstate) by primitive undifferentiated mesenchyme cells. This stareinvolves the production of chondrocyte-specific markers without theability to produce a typical cartilage ECM. Subsequently, the cellsdevelop the capacity to produce a cartilage-specific ECM as theydifferentiate into chondrocytes. Langille, Microscop. Res. & Tech.28:455-469 (1994). Chondrocyte migration, adhesion, and proliferationthen contribute to the development of bony. and cartilaginous, skeleton.Abnormal elaboration of the programmed development of cellsparticipating in the process of chondrogenesis results in skeletaldefects presenting problems that range from cosmetic concerns tolife-threatening disorders.

[0008] Like angioogenesis and chondrogenesis, oncogenesis ischaracterized by changes in cell adhesion, migration. and proliferation.Metastasizing cancer cells exhibit altered adhesion and migrationproperties. Establishment of tumorous masses requires increased cellproliferation and the elaboration of the cellular propertiescharacteristic of angiogenesis during the neovascularization of tumors.

[0009] Abnormal progression of angiogenesis or chondrogenesis, as wellas mere progression of oncogenesis, substantially impairs the quality oflife for afflicted individuals and adds to modern health care costs. Thefeatures common to these complex biological processes. comprisingaltered cell adhesion, migration, and proliferation, suggest that agentscapable of influencing all three of these cellular activities would beeffective in screening for, and modulating, the aforementioned complexbiological processes. Although the art is aware of agents that influenceindividual cellular activities. e.g., integrins and selectins (celladhesion), chemokines (cell migration). and a variety of growth factorsor cytokines (cell proliferation), until recently no agent has beenidentified that exerts an influence over all three cellular activitiesin humans.

[0010] Murine Cyr61 (CYsteine-Rich protein) is a protein expressed inactively growing and dividing cells that may influence each of thesethree cellular activities. RNase protection analyses have shown that thegene encoding murine Cyr61, murine Cyr61, is transcribed in thedeveloping mouse embryo. O'Brien et al., Cell Growth & Diff 3:645-654(1992). In situ hybridization analysis showed that expression of cyr61during mouse embryogenesis is closely correlated with thedifferentiation of mesenchymal cells. derived from ectoderm andmesoderm, into chondrocytes. In addition, cyr61 is expressed in thevessel walls of the developing circulatory system. These observationsindicate that murine cyr61 is expressed during cell proliferation anddifferentiation, which are characteristics of expression of genesinvolved in regulatory cascades that control the cell growth cycle.

[0011] Further characterization of the Cyr61 polypeptide has beenhampered by an inability to purify useful quantities of the protein.Efforts to purify Cyr61 in quantity by overexpression from eithereukaryotic or prokaryotic cells typically fail. Yang, University ofIllinois at Chicago, Ph.D. Thesis (1993). One problem associated withattempting to obtain useful quantities of Cyr61 is the reduction inmammalian growth rates induced by overexpression of Cyr61. Anotherproblem with Cyr61 purification is that the cysteine-rich polypeptide,when expressed in bacterial cells using recombinant DNA techniques, isoften found in insoluble protein masses. Nevertheless. Cyr61 has beencharacterized as a polypeptide of 349 amino acids, containing 39cysteine residues, a hydrophobic putative N-terminal signal sequence,and potential N-linked glycosylation sites (Asn₂₈ and Asn₂₂₅). U.S. Pat.No. 5,408,040 at column 3, lines 41-54, Grotendorst et al. incorporatedherein by reference (the '040 Patent).

[0012] Recently, proteins related to Cyr61 have been characterized.

[0013] For example, a human protein, Connective Tissue Growth Factor(CTGF). has been identified. (See '040 Patent). CTGF is expressed inactively growing cells such as fibroblasts and endothelial cells ('040Patent. at column 5. lines 62-64). an expression pattern shared byCyr61. In terms of function. CTGF has been described as a protein growthfactor because its primary biological activity has been alleged to beits mitogenicity ('040 Patent, at column 2, lines 25-27 and 53-55). Inaddition, CTGF reportedly exhibits chemotactic activity. '040 Patent, atcolumn 2. lines 56-59. In terms of structure. the polynucleotidesequence encoding CTGF, and the amino acid sequence of CTGF. have beenpublished. '040 Patent, SEQ ID NO: 7 and SEQ ID NO: 8. respectively.

[0014] Another apparently related protein is the mouse protein Fisp12(FIbroblast Secreted Protein). Fisp12 has been subjected to amino acidsequence analysis, revealing a primary structure that is rich incysteines. Ryseck et al., Cell Growth & Diff 2:225-233 (1991),incorporated herein by reference. The protein also possesses ahydrophobic N-terminal sequence suggestive of the signal sequencecharacteristic of secreted proteins.

[0015] Sequence analyses involving Cyr61, Fisp12, CTGF, and otherproteins, have contributed to the identification of a family ofcysteine-rich secreted proteins. Members of the family share similarprimary structures encoded by genes exhibiting similar sequences. Eachof the proteins in this emerging family is further characterized by thepresence of a hydrophobic N-terminal signal sequence and 38 cysteineresidues in the secreted forms of the proteins. Members of the familyidentified to date include the aforementioned Cyr61 (human and mouse),Fisp12 (mouse), and CTGF (the human ortholog of Fisp12), as well asCEF10 (chicken), and Nov (avian).

[0016] One of several applications for a purified protein able to affectcell adhesion, migration, and proliferation properties involves thedevelopment of stable, long term ex vivo hematopoietic stem cellcultures. Patients subjected to high-dose chemotherapy have suppressedhematopoiesis: expansion of stem cells, their maturation into varioushematopoietic lineages. and mobilization of mature cells intocirculating blood routinely take many weeks to complete. For suchpatients, and others who need hematopoietic cell transplantation.introduction into those patients of autologous stem cells that have beenmanipulated and expanded in culture is advantageous. Such hematopoieticstem cells (HSC) express the CD34 stem cell antigen, but do not expresslineage commitment antigens. These cells can eventually give rise to allblood cell lineages (e.g., erythrocytes, lymphocytes, and myelocytes).

[0017] Hematopoietic progenitor cells that can initiate and sustain longterm cultures (i.e. long term culture system-initiating cells or LTC-IC)represent a primitive population of stem cells. The frequency of LTC-IChas been estimated at only 1-2 per 10⁴ cells in normal human marrow andonly about 1 per 50-100 cells in a highly purified CD34⁺ subpopulation.Thus, it would be useful to have methods and systems for long term cellculture that maintain and expand primitive, pluripotent human HSC to beused for repopulation of the hematopoietic system in vivo.

[0018] Cell culture models of hematopoiesis have revealed a multitude ofcytokines that appear to play a role in the hematopoietic process,including various colony stimulating factors. interletukins, stem cellfactor, and the c-kit ligand. However, in ex vivo cultures, differentcombinations of these cytokines favor expansion of different sets ofcommitted progenitors. For example, a factor in cord blood plasmaenhanced expansion of granulocyte-erythroid-macrophage-megakaryocytecolony forming unit (CFU-GEMM) progenitors, but expansion in thesecultures favored the more mature subsets of cells. Therefore, it hasbeen difficult to establish a culture system that mimics in vivohematopoiesis.

[0019] An HSC culture system should maintain and expand a large numberof multi- or pluripotent stem cells capable of both long termrepopulation and eventual lineage commitment under appropriateinduction. However, in most ex vivo culture systems, the fraction of thecell population comprised of LTC-IC decreases steadily with continuedculturing, often declining to 20% of their initial level after severalweeks. as the culture becomes populated by more mature subsets ofhematopoietic progenitor cells that are no longer pluripotent. Moreover,the proliferative capacity exhibited by individual LTC-IC may varyextensively. Thus, a need exists in the art for HSC culture systemscomprising biological agents that maintain or promote the pluripotentpotential of cells such as LTC-IC cells. In addition to a role indeveloping ex vivo HSC cultures, biological agents affecting celladhesion, migration, and proliferation are useful in a variety of othercontexts.

[0020] Proteins that potentiate the activity of mitogens but have nomitogenic activity themselves may play important roles as signallingmolecules in such processes as hematopoiesis. Moreover, these signallingproteins could also serve as probes in the search for additionalmitogens, many of which have not been identified or characterized.Several biological factors have been shown to potentiate the mitogenicactivity of other factors. without being mitogenic themselves. Some ofthese potentiators are associated with the cell surface and/orextracellular matrix. Included in this group are a secreted basicFibroblast Growth Factor-binding protein (bFGF-binding protein), thebasal lamina protein perlecan. and the Human Immunodeficiency Virus-1TAT protein, each protein being able to promote bFGF-induced cellproliferation and angiogenesis. Also included in this group of mitogenpotentiators are thrombospondin, capable of activating a latent form ofTransforming Growth Factor-β, and an unidentified secretedgrowth-potentiating factor from vascular smooth muscle cells (Nakano etal., J. Biol. Chem. 270:5702-5705 [1995]), the latter factor beingrequired for efficient activation of Epidermal Growth Factor- orthrombin-induced DNA synthesis. Further, the B cell stimulatoryfactor-1/interleukin-4, a T cell product with no demonstrable mitogenicactivity, is able to 1) enhance the proliferative response ofgranulocyte-macrophage progenitors to granulocyte-colony stimulatingfactor. 2) enhance the proliferative response of erythroid progenitorsto erythropoietin. and 3) together with erythropoietin, induce colonyformation by multipotent progenitor cells. Similarly, interleukin-7enhanced stem cell factor-induced colony formation by primitive murinebone marrow progenitors. although interleukin-7 had no proliferativeeffect by itself. In addition, lymphocyte growth enhancing factor (LGEF)was found to enhance mitogen-stimulated human peripheral bloodlymphocyte (PBL) or purified T cell proliferation in a dose-dependentfashion. LGEF alone did not stimulate PBL or T cell proliferation.

[0021] Therefore. a need continues to exist for biological agentscapable of exerting a concerted and coordinated influence on one or moreof the particularized functions collectively characterizing such complexbiological processes as angiogenesis. chondrogenesis, and oncogenesis.In addition, a need persists in the art for agents contributing to thereproduction of these in vivo processes in an ex vivo environment, e.g.,the development of HSC cultures. Further. there continues to be a needfor tools to search for the remaining biological components of thesecomplex processes, e.g., mitogen probes, the absence of which impedesefforts to advantageously modulate and thereby control such processes.

SUMMARY OF THE INVENTION

[0022] The present invention provides extracellular matrix (ECM)signalling molecule-related materials and methods. In particular. thepresent invention is directed to polynucleotides encoding ECM signallingmolecules and fragments or analogs thereof, ECM signallingmolecule-related polypeptides and fragments, analogs, and derivativesthereof, methods of producing ECM signalling molecules, and methods ofusing ECM signalling molecules.

[0023] One aspect of the present invention relates to a purified andisolated polypeptide comprising an ECM signalling molecule. Thepolypeptides according to the invention retain at least one biologicalactivity of an ECM signalling molecule, such as the ability to stimulatecell adhesion. cell migration, or cell proliferation; the ability tomodulate angiogenesis. chondrogenesis. or oncogenesis: immunogenicity orthe ability to elicit an immune response, and the ability to bind topolypeptides having specific binding sites for ECM signalling moleculesincluding antibodies and integrins. The polypeptides may be native orrecombinant molecules. Further, the invention comprehends full-lengthECM signalling molecules, and fragments thereof. In addition, thepolypeptides of the invention may be underivatized, or derivatized inconformity with a native or non-native derivatization pattern. Theinvention further extends to polypeptides having a native or naturallyoccurring amino acid sequence, and variants (i.e., polypeptides havingdifferent amino acid sequences), analogs (i.e., polypeptides having anon-standard amino acid or other structural variation from theconventional set of amino acids) and homologs (i.e., polypeptidessharing a common evolutionary ancestor with another polypeptide)thereof. Polypeptides that are covalently linked to other compounds,such as polyethylene glycol, or other proteins or peptides, i.e. fusionproteins, are contemplated by the invention.

[0024] Exemplary ECM signalling molecules include mammalian Cyr61,Fisp12. and CTGF polypeptides. Beyond ECM signalling molecules. theinvention includes polypeptides that specifically bind an ECMsignallinig molecule of the invention, such as the aforementionedantibody products. A wide variety of antibody products fall within thescope of the invention. including polyclonal and monoclonal antibodies,antibody fragments, chimeric antibodies. CDR-grafted antibodies,“humanized” antibodies, and other antibody forms known in the art. Othermolecules such as peptides, carbohydrates or lipids designed to bind toan active site of the ECM molecules thereby inhibiting their activitiesare also contemplated by the invention. However molecules such aspeptides that enhance or potentiate the activities of ECM molecule arealso within the scope of the invention. The invention further extends toa pharmaceutical composition comprising a biologically effective amountof a polypeptide and a pharmaceutically acceptable adjuvant, diluent orcarrier, according to the invention. A “biologically effective amount”of the biomaterial is an amount that is sufficient to result in adetectable response in the biological sample when compared to a controllacking the biomaterial.

[0025] Another aspect of the invention relates to a purified andisolated polynucleotide comprising a sequence that encodes a polypeptideof the invention. A polynucleotide according to the invention may be DNAor RNA, single- or double-stranded. and may be may purified and isolatedfrom a native source, or produced using synthetic or recombinanttechniques known in the art. The invention also extends topolynucleotides encoding fragments, analogs (i.e., polynucleotideshaving a non-standard nucleotide), homologs (i.e., polynucleotideshaving a common evolutionary ancestor with another polynucleotide).variants (i.e., polynucleotides differing in nucleotide sequence), andderivatives (i.e., polynucleotides differing in a structural manner thatdoes not involve the primary nucleotide sequence) of ECM molecules.Vectors comprising a polynucleotide according to the invention are alsocontemplated. In addition, the invention comprehends host cellstransformed or transfected with a polynucleotide or vector of theinvention.

[0026] Other aspects of the invention relate to methods for making orusing the polypeptides and/or polynucleotides of the invention. A methodfor making a polypeptide according to the invention comprises expressinga polynucleotide encoding a polypeptide according to the presentinvention in a suitable host cell and purifying the polypeptide. Othermethods for making a polypeptide of the invention use techniques thatare known in the art such as the isolation and purification of nativepolypeptides or the use of synthetic techniques for polypeptideproduction. In particular, a method of purifying an ECM signallingmolecule such as human Cyr61 comprises the steps of identifying a sourcecontaining human Cyr61, exposing the source to a human Cyr61-specificbiomolecule that binds Cyr61 such as an anti-human Cyr61 antibody, andeluting the human Cyr61 from the antibody or other biomolecule. therebypurifying the human Cyr61.

[0027] Another aspect of the invention is a method of screening for amodulator of angiogenesis comprising the steps of: (a) contacting afirst biological sample capable of undergoing angiogenesis with abiologically effective (i.e., angiogenically effective) amount of an ECMsignalling molecule-related biomaterial and a suspected modulator(inhibitor or potentiator); (b) separately contacting a secondbiological sample with a biologically effective amount of an ECMsignalling molecule-related biomaterial. thereby providing a control:(c) measuring the level of angiogenesis resulting from step (a) and fromstep (b); and (d) comparing the levels of angiogenesis measured in step(c), whereby a modulator of angiogenesis is identified by its ability toalter the level of angiogenesis when compared to the control of step(b). The modulator may be either a potentiator or inhibitor ofangiogenesis and the ECM signalling molecule-related biomaterialincludes. but is not limited to, Cyr61, and fragments, variants,homologs, analogs, derivatives, and antibodies thereof.

[0028] The invention also extends to a method of screening for amodulator of angiogenesis comprising the steps of: (a) preparing a firstimplant comprising Cyr61 and a second implant comprising Cyr61 and asuspected modulator of Cyr61 angiogenesis; (b) implanting the firstimplant in a first cornea of a test animal and the second implant in asecond cornea of the test animal: (c) measuring the development of bloodvessels in the first and second corneas: and (d) comparing the levels ofblood vessel development measured in step (c). whereby a modulator ofangiogenesis is identified by its ability to alter the level of bloodvessel development in the first cornea when compared to the blood vesseldevelopment in the second cornea.

[0029] Another aspect of the invention relates to a method of screeningfor a modulator of chondrogenesis comprising the steps of: (a)contacting a first biological sample capable of undergoingchondrogenesis with a biologically effective (e.g. chondrogenicallyeffective) amount of an ECM signalling molecule-related biomaterial anda suspected modulator; (b) separately contacting a second biologicalsample capable of undergoing chondrogenesis with a biologicallyeffective amount of an ECM signalling molecule-related biomaterial,thereby providing a control; (c) measuring the level of chondrogenesisresulting from step (a) and from step (b); and (d) comparing the levelsof chondrogenesis measured in step (c), whereby a modulator ofchondrogenesis is identified by its ability to alter the level ofchondrogenesis when compared to the control of step (b). The modulatormay be either a promoter or an inhibitor of chondrogenesis; the ECMsignalling molecules include those defined above and compounds such asmannose-6-phosphate, heparin, and tenascin.

[0030] The invention also relates to an in vitro method of screening fora modulator of oncogenesis comprising the steps of: (a) inducing a firsttumor and a second tumor; (b) administering a biologically effectiveamount of an ECM signalling molecule-related biomaterial and a suspectedmodulator to the first tumor; (c) separately administering abiologically effective amount of an ECM signalling molecule-relatedbiomaterial to the second tumor, thereby providing a control; (d)measuring the level of oncogenesis resulting from step (b) and from step(c); and (e) comparing the levels of oncogenesis measured in step (d),whereby a modulator of oncogenesis is identified by its ability to alterthe level of oncogenesis when compared to the control of step (c).Modulators of oncogenesis contemplated by the invention includeinhibitors of oncogenesis. Tumors may be induced by a variety oftechniques including. but not limited to, the administration ofchemicals, e.g., carcinogens. and the implantation of cancer cells. Arelated aspect of the invention is a method for treating a solid tumorcomprising the step of delivering a therapeutically effective amount ofa Cyr61 inhibitor to an individual, thereby inhibiting theneovascularization of the tumor. Inhibitors include, but are not limitedto. inhibitor peptides such as peptides having the “RGD” motif, andcytotoxins, which may be free or attached to molecules such as Cyr61.

[0031] Yet another aspect of the invention is directed to a method ofscreening for a modulator of cell adhesion comprising the steps of: (a)preparing a surface compatible with cell adherence; (b) separatelyplacing first and second biological samples capable of undergoing celladhesion on the surface; (c) contacting a first biological sample with asuspected modulator and a biologically effective amount of an ECMsignalling molecule-related biomaterial selected from the groupconsisting of a human Cyr61, a human Cyr61 fragment, a human Cyr61analog, and a human Cyr61 derivative; (d) separately contacting a secondbiological sample with a biologically effective amount of an ECMsignalling molecule-related biomaterial selected from the groupconsisting of a human Cyr61, a human Cyr61 fragment, a human Cyr61analog, and a human Cyr61 derivative, thereby providing a control; (e)measuring the level of cell adhesion resulting from step (c) and fromstep (d); and (f) comparing the levels of cell adhesion measured in step(e), whereby a modulator of cell adhesion is identified by its abilityto alter the level of cell adhesion when compared to the control of step(d).

[0032] The invention also extends to a method of screening for amodulator of cell migration comprising the steps of: (a) forming a gelmatrix comprising Cyr61 and a suspected modulator of cell migration: (b)preparing a control gel matrix comprising Cyr61; (c) seeding endothelialcells capable of undergoing cell migration onto the gel matrix of step(a) and the control gel matrix of step (b); (d) incubating theendothelial cells; (e) measuring the levels of cell migration byinspecting the interior of the gel matrix and the control gel matrix forcells: (f) comparing the levels of cell migration measured in step (e).whereby a modulator of cell migration is identified by its ability toalter the level of cell migration in the gel matrix when compared to thelevel of cell migration in the control gel matrix. The endothelial cellsinclude, but are not limited to, human cells, e.g., human microvascularendothelial cells. The matrix may be formed from gelling materials suchas Matrigel, collagen. or fibrin or combinations thereof.

[0033] Another aspect of the invention is directed to an in vitro methodof screening for cell migration comprising the steps of: (a) forming afirst gelatinized filter and a second gelatinized filter, each filterhaving two sides; (b) contacting a first side of each the filter withendothelial cells, thereby adhering the cells to each the filter: (c)applying an ECM signalling molecule and a suspected modulator of cellmigration to a second side of the first gelatinized filter and an ECMsignalling molecule to a second side of the second gelatinized filter;(d) incubating each the filter; (e) detecting cells on the second sideof each the filter, and (f) comparing the presence of cells on thesecond side of the first gelatinized filter with the presence of cellson the second side of the second gelatinized filter, whereby a modulatorof cell migration is identified by its ability to alter the level ofcell migration measured on the first gelatinized filter when compared tothe cell migration measured on the second gelatinized filter. Theendothelial cells are defined above. The ECM signalling molecules extendto human Cyr61 and each of the filters may be placed in apparatus suchas a Boyden chamber. including modified Boyden chambers.

[0034] The invention also embraces an in vivo method of screening for amodulator of cell migration comprising the steps of: (a) removing afirst central portion of a first biocompatible sponge and a secondcentral portion of a second biocompatible sponge; (b) applying an ECMsignalling molecule and a suspected modulator to the first centralportion and an ECM signalling molecule to the second central portion;(c) reassociating the first central portion with said firstbiocompatible sponge and said second central portion with the secondbiocompatible sponge; (d) attaching a first filter to a first side ofthe first biocompatible sponge and a second filter to a second side ofthe first biocompatible sponge; (e) attaching a third filter to a firstside of the second biocompatible sponge and a fourth filter to a secondside of the second biocompatible sponge; (f) implanting each of thebiocompatible sponges, each biocompatible sponge comprising the centralportion and the filters, in a test animal; (e) removing each the spongefollowing a period of incubation; (f) measuring the cells found withineach of the biocompatible sponges; and (g) comparing the presence ofcells in the first biocompatible sponge with the presence of cells inthe second biocompatible sponge, whereby a modulator of cell migrationis identified by its ability to alter the level of cell migrationmeasured using the first biocoinpatible sponge when compared to the cellmigration measured using the second biocompatible sponge. ECM signallingmolecules include. but are not limited to. human Cyr61; the ECMsignalling molecule may also be associated with Hydron. In addition, thein vivo method of screening for a modulator of cell migration mayinclude the step of providing a radiolabel to the test animal anddetecting the radiolabel in one or more of the sponges.

[0035] Another aspect of the invention relates to a method formodulating hemostasis comprising the step of administering an ECMsignalling molecule in a pharmaceutically acceptable adjuvant, diluentor carrier. Also, the invention extends to a method of inducing woundhealing in a tissue comprising the step of contacting a wounded tissuewith a biologically effective amount of an ECM signalling molecule.thereby promoting wound healing. The ECM signalling molecule may beprovided in the form of an ECM signalling molecule polypeptide or an ECMsignalling molecule nucleic acid, e.g., using a gene therapy technique.For example, the nucleic acid may comprise an expression controlsequence operably linked to an ECM signalling molecule which is thenintroduced into the cells of a wounded tissue. The expression of thecoding sequence is controlled, e.g., by using a tissue-specific promotersuch as the K14 promoter operative in skin tissue to effect thecontrolled induction of wound healing. The nucleic acid may include avector such as a Herpesvirus. an Adenovirus. an Adeno-associated Virus,a Cytomegalovirus, a Baculovirus, a retrovirus, and a Vaccinia Virus.Suitable wounded tissues for treatment by this method include, but arenot limited to, skin tissue and lung epithelium. A related methodcomprises administering a biologically effective amount of an ECMsignalling molecule. e.g. Cyr61, to an animal to promote organregeneration. The impaired organ may be the result of trauma, e.g.surgery, or disease. Another method of the invention relates toimproving the vascularization of grafts. e.g., skin grafts. Anothermethod of the invention is directed to a process for promoting boneimplantation, including bone grafts. The method for promoting boneimplantation comprises the step of contacting a bone implant orreceptive site with a biologically effective (i.e., chondrogenicallyeffective) amount of an ECM signalling molecule. The contacting step maybe effected by applying the ECM signalling molecule to a biocompatiblewrap such as a biodegradable gauze and contacting the wrap with a boneimplant, thereby promoting bone implantation. The bone implants comprisenatural bones and fragments thereof, as well as inanimate natural andsynthetic materials that are biocompatible, such as prostheses. Inaddition to direct application of an ECM signalling molecule to a bone,prosthesis, or receptive site, the invention contemplates the use ofmatrix materials for controlled release of the ECM signalling molecule,in addition to such application materials as gauzes.

[0036] Yet another aspect of the invention relates to a method ofscreening for a modulator of cell proliferation comprising the steps of:(a) contacting a first biological sample capable of undergoing cellproliferation with a suspected modulator and a biologically effective(i.e., mitogenically effective) amount of an ECM signallingmolecule-related biomaterial selected from the group consisting of ahuman Cyr61, a human Cyr61 fragment, a human Cyr61 analog, and a humanCyr61 derivative; (b) separately contacting a second biological samplecapable of undergoing cell proliferation with a biologically effectiveamount of an ECM signalling molecule-related biomaterial selected fromthe group consisting of a human Cyr61, a human Cyr61 fragment, a humanCyr61 analog, and a human Cyr61 derivative, thereby providing a control;(c) incubating the first and second biological samples; (d) measuringthe level of cell proliferation resulting from step (c); and (e)comparing the levels of cell proliferation measured in step (d), wherebya modulator of cell proliferation is identified by its ability to alterthe level of cell adhesion when compared to the control of step (b).

[0037] Also comprehended by the invention is a method for expanding apopulation of undifferentiated hematopoietic stein cells in culture,comprising the steps of: (a) obtaining hematopoietic stem cells from adonor: and (b) culturing said cells under suitable nutrient conditionsin the presence of a biologically effective (i.e., hematopoieticallyeffective) amount of Cyr61.

[0038] Another method according to the invention is a method ofscreening for a mitogen comprising the steps of: (a) plating cellscapable of undergoing cell proliferation: (b) contacting a first portionof the cells with a solution comprising Cyr61 and a suspected mitogen;(c) contacting a second portion of the cells with a solution comprisingCyr61, thereby providing a control; (c) incubating the cells; (d)detecting the growth of the first portion of cells and the secondportion of the cells; and (e) comparing growth of the first and secondportions of cells, whereby a mitogen is identified by its ability toinduce greater growth in the first portion of cells when compared to thegrowth of the second portion of cells. The cells include, but are notlimited to. endothelial cells and fibroblast cells. Further, the methodmay involve contacting the cells with a nucleic acid label. e.g.,[³H]-thymidine. and detecting the presence of the label in the cells.Another method relates to improving tissue grafting. comprisingadministering to an animal a quantity of Cyr61 effective in improvingthe rate of neovascularization of a graft.

[0039] Numerous additional aspects and advantages of the presentinvention will be apparent upon consideration of the following drawingand detailed description.

BRIEF DESCRIPTION OF THE DRAWING

[0040]FIG. 1 presents the comparative amino acid sequences of members ofthe cysteine-rich protein family of growth-regulating proteins.

DETAILED DESCRIPTION OF THE INVENTION

[0041] In the mouse, the Cyr61 protein has been found to influence celladhesion, migration, and proliferation. The cyr61 gene. which encodesCyr61, is an immediate-early gene that is transcriptionally activated byserum growth factors in mouse fibroblasts. Lau et al., EMBO J.4:3145-3151 (1985). incorporated herein by reference; Lau et al., Proc.Natl. Acad. Sci. (USA) 84:1182-1186 (1987), incorporated herein byreference. The murine cyr61 cDNA coding sequence is set forth in SEQ IDNO: 1. (The human cyr61 cDNA coding sequence is provided in SEQ ID NO:3). The amino acid sequence of murine Cyr61 is set out in SEQ ID NO: 2.(The human Cyr61 amino acid sequence is presented in SEQ ID NO: 4).Cyr61 is a 41 kDa polypeptide exhibiting 39 cysteine residues,approximately 10% of the 379 amino acids constituting the unprocessedprotein. Yang et al., Cell Growth & Diff 2:351-357 (1991), incorporatedherein by reference. Investigations have revealed that murine Cyr61binds heparin and is secreted. Yang et al. Consistent with the observedsecretion of Cyr61 is the identification of an N-terminal signalsequence in nascent Cyr61, deduced from inspection of the murine cyr61cDNA sequence. Yang et al. Additionally, Cyr61 is not found in theconditioned medium of cultured cells expressing Cyr61, but is foundassociated with the extracellular matrix (ECM) and the cell surface.Yang et al. Structurally similar cysteine-rich mammalian proteins havebeen characterized.

[0042] Fisp12. a cysteine-rich murine protein, exhibits structuralsimilarity to Cyr61. The cDNA sequence encoding Fisp12 is set forth inSEQ ID NO: 5; the amino acid sequence of Fisp12 is presented in SEQ IDNO: 6. Murine Fisp12, like Cyr61. influences cell adhesion,proliferation and migration. The human ortholog of Fisp12 is ConnectiveTissue Growth Factor (CTGF), a protein similar in structure and functionto Cyr61. Fisp12, and CTGF. are distinguishable from Cyr61, however. Forexample, a greater proportion of secreted Fisp12 is found in the culturemedium than is the case with Cyr61, a correspondingly lower proportionof Fisp12 is localized in the area of expressing cells (cell surface andnearby extracellular matrix) than is found with Cyr61. Additionalsimilarities and distinctions among the proteins comprising the ECMsignalling molecules of the invention will become apparent in therecitations hereinbelow.

[0043] The present invention has multiple aspects. illustrated by thefollowing examples. Example 1 describes the cloning of polynucleotidesencoding members of the cysteine-rich protein family of ECM signallingmolecules: Example 2 describes sequence analyses; Example 3 describesRNA analyses; Example 4 describes the production of transgenic animals;Example 5 describes the expression of Cyr61 polypeptides: Example 6describes the expression of Fisp12 polypeptides; Example 7 sets outmethods of polypeptide purification: Example 8 provides acharacterization of the polypeptides of the invention; Example 9discloses a heparin binding assay for the polypeptide members of thecysteine-rich protein family: Example 10 is directed to receptors forthe polypeptides: Example 11 describes anti-ECM signalling moleculeantibodies: Example 12 is directed to inhibitory peptides: Example 13describes cell adhesion and polypeptide-based methods for influencingthe process of cell adhesion: Example 14 describespolypeptide-influenced migration of fibroblasts: Example 15 describesthe migration of endothelial cells and in vitro assays for migration:Example 16 describes an in vitro assay for inhibitors of endothelialcell migration: Example 17 describes an in vivo assay for endothelialcell migration: Example 18 describes mitogen potentiation by thepolypeptides of the invention; Example 19 describes an in vivo corneaassay for angiogenic factors and modulators; Example 20 is directed tomethods for influencing blood clotting using the polypeptides of theinvention; Example 21 discloses the use of the polypeptides for ex vivohematopoietic stem cell cultures: Example 22 addresses organregeneration; Example 23 describes chondrogenesis and the expression ofextracellular matrix signalling molecules in mesenchyme cells; Example24 describes the promotion of cell adhesion in the process ofchondrogenesis using the polypeptides of the invention; Example 25describes chondrogenesis and the influence of the polypeptides of theinvention on cell aggregation: Example 26 describes the promotion ofcell proliferation by polypeptides of the invention in the process ofchondrogenesis; Example 27 addresses methods for using the polypeptidesof the invention to affect chondrogenesis; and Example 28 providesgenetic approaches to the use of polynucleotides of the invention. Theseexamples are intended to be illustrative of the present invention andshould not be construed to limit the scope of the invention.

EXAMPLE 1 Polynucleotide Cloning

[0044] A human cyr61 cDNA was isolated from a human placental cDNAlibrary by probing with the murine cyr61cDNA sequence using techniquesthat are standard in the art. See Sambrook et al., incorporated hereinby reference. Isolation of the complete murine cyr61cDNA from a BALB/c3T3 (ATCC CRL-1658) cDNA library has been described. O'Brien et al.,Mol. Cell. Biol. 10:3569-3577 (1990), incorporated herein by reference.The nucleotide and deduced amino acid sequences of murine cyr61 areavailable from the GenBank database under accession number M32490. Thenucleotide sequence of murine cyr61is presented in SEQ ID NO: 1; themurine Cyr61 amino acid sequence is presented in SEQ ID NO: 2.

[0045] The human cDNA library was constructed using λgt11 (PromegaCorp., Madison. Wis.) as a vector which was transfected into E. coli andplated on LB agar. A murine cDNA expression construct cloned in pGEM-2(O'Brien et al., [1990]), containing the entire murine cyr61 codingsequence [nucleotides 56-1560, using the numbering of O'Brien et al.,(1990); see SEQ ID NO: 1] was used as a probe. The mouse cDNA probe wasradiolabeled by techniques standard in the art. Sambrook et al. Plaquescreenings using the mouse probe were performed using standardtechniques.

[0046] Sambrook et al.

[0047] More particularly, agar plates containing the human cDNA librarydescribed above were exposed to nitrocellulose filters (BA85, 82 mm,Schleicher & Schuell, Keene, N.H.) were placed on each plate. Afterplaque adsorption (approximately 20 minutes), the filters were removedand air dried for approximately 30 minutes. Subsequently, each filterwas sequentially submerged for 30-60 seconds in 0.2 M NaOH, 1.5 M NaCl(100 ml); 2×SSC, 0.4 M Tris-HCl, pH 7.4 (100 ml); and 0.2×SSC (100 ml).Filters were then dried at room temperature for approximately 1 hour andsubjected to 80° C. under vacuum for 2 hours. Filters were probed withradiolabeled murine cyr61 cDNA.

[0048] Alternatively, human cyr61 cDNA clones were identified withprobes generated by RT-PCR. In particular, the probe for screening thehuman placental cDNA library was a PCR fragment generated withdegenerate primers by RT-PCR of total RNA from logarithmically growingWI38 cells. The primers were derived from the sequences corresponding tothe most conserved region of the open reading frame of the mousecyr61cDNA. One primer. designated H61-5 [5′-GGGAATTCTG(TC)GG(GATC)TG(TC)T-G(TC)AA(GA)GT(GC)TG-3′], contains a degenerate sequence which, withthe exception of the “GGGAATTC” sequence at the 5′ end which was used tointroduce an EcoRI site. is derived from nucleotides 327-346 (sensestrand) of the mouse cyr61 sequence set forth in SEQ ID NO: 1. Thedegeneracies appear in positions corresponding to the third position ofcodons in SEQ ID NO: 1. The second primer used for PCR amplification ofa human cyr61 sequence was designated H61-3 [5′-CCGGATCC(GA)CA(GA)TT(GA) T-A(GA)Tr(GA)CA-3′], which. with theexception of the 5′ sequence “CCGGATCC” used to introduce a BamHI site,corresponds to the anti-sense strand complementary to nucleotides1236-1250 of the mouse cyr61 sequence set forth in SEQ ID NO: 1. Thedegeneracies occur in positions complementary to the third positions ofcodons in mouse cyr61 as set forth in SEQ ID NO: 1. The amplifiedcyr61cDNA was cloned into the pBlueScript SK+vector (Stratagene, LaJolla, Calif.) and sequenced with a Sequenase II kit (U.S. Biochemicals,Cleveland, Ohio.).

[0049] Serial screenings of the human placental cDNA library led to theisolation of a clone containing a human cyr61 cDNA. The human cir61 cDNAis approximately 1,500 bp in length. The human cDNA is contained on anEcoRI fragment cloned into the EcoRI site in pGEM-2. As shown in SEQ IDNO: 3. the human cDNA sequence includes the entire coding region forhuman Cyr61. along with 120 bp of 5′ flanking sequence, and about 150 bpof 3′ flanking sequence.

[0050] The polynucleotides of the invention may be wholly or partiallysynthetic. DNA or RNA, and single- or double-stranded. Becausepolynucleotides of the invention encode ECM signalling moleculepolypeptides which may be fragments of an ECM signalling moleculeprotein, the polynucleotides may encode a partial sequence of an ECMsignalling molecule. Polynucleotide sequences of the invention areuseful for the production of ECM signalling molecules by recombinantmethods and as hybridization probes for polynucleotides encoding ECMsignalling molecules.

[0051] DNA polynucleotides according to the invention include genomicDNAs. cDNAs, and oligonucleotides comprising a coding sequence of an ECMsignalling molecule, or a fragment or analog of an ECM signallingmolecule, as described above. that retains at least one of thebiological activities of an ECM signalling molecule such as the abilityto promote cell adhesion, cell migration, or cell proliferation in suchbiological processes as angiogenesis, chondrogenesis, and oncogenesis,or the ability to elicit an antibody recognizing an ECM signallingmolecule.

[0052] Other polynucleotides according to the invention differ insequence from sequences contained within native ECM signalling moleculepolynucleotides (i.e., by the addition, deletion, insertion, orsubstitution of nucleotides) provided the polynucleotides encode aprotein that retains at least one of the biological activities of an ECMsignalling molecule. A polynucleotide sequence of the invention maydiffer from a native ECM signalling molecule polynucleotide sequence bysilent mutations that do not alter the sequence of amino acids encodedtherein. Additionally. polynucleotides of the invention may specify anECM signalling molecule that differs in amino acid sequence from nativeECM signalling molecule sequences or subsequences, as described above.For example, polynucleotides encoding polypeptides that differ in aminoacid sequence from native ECM signalling molecules by conservativereplacement of one or more amino acid residues. are contemplated by theinvention. The invention also extends to polynucleotides that hybridizeunder standard stringent conditions to polynucleotides encoding an ECMsignalling molecule of the invention. or that would hybridize but forthe degeneracy of the genetic code. Exemplary stringent hybridizationconditions involve hybridization at 42° C. in 50% formamide, 5×SSC. 20mM Na•PO₄. pH 6.8 and washing in 0.2×SSC at 55° C. It is understood bythose of skill in the art that variation in these conditions occursbased on the length and GC nucleotide content of the sequences to behybridized. Formulas standard in the art are appropriate for determiningexact hybridization conditions. See Sambrook et al., Molecular Cloning:A Laboratory Manual (Second ed. Cold Spring Harbor Laboratory Press1989) §§ 9.47-9.51.

[0053] ECM signalling molecule polynucleotides comprising RNA are alsowithin the scope of the present invention. A preferred RNApolynucleotide according to the invention is an mRNA of human cyr61.Other RNA polynucleotides of the invention include RNAs that differ froma native ECM signalling molecule mRNA by the insertion, deletion,addition, or substitution of nucleotides (see above), with the provisothat they encode a polypeptide retaining a biological activityassociated with an ECM signalling molecule. Still other RNAs of theinvention include anti-sense RNAs (i.e., RNAs comprising an RNA sequencethat is complementary to an ECM signalling molecule mRNA).

[0054] Accordingly, in another embodiment a set of DNA fragmentscollectively spanning the human cyr61 cDNA were cloned in pGEM-2 and M13derivatives using methods well known in the art to facilitate nucleotidesequence analyses. The pGEM-2 clones provided substrates for theenzymatic generation of serial deletions using techniques known in theart. This collection of clones, collectively containing a series of DNAfragments spanning various parts of the cyr61cDNA coding region, areuseful in the methods of the invention. The resulting series of nestedpGEM-2 clones, in turn, provided substrates for nucleotide sequenceanalyses using the enzymatic chain terminating technique. The fragmentsare also useful as nucleic acid probes and for preparing Cyr61 deletionor truncation analogs. For example, the cyr61 cDNA clones may be used toisolate cyr61 clones from human genomic libraries that are commerciallyavailable. (Clontech Laboratories. Inc. Palo Alto, Calif.). Genomicclones. in turn, may be used to map the cyr61 locus in the human genome,a locus that may be associated with a known disease locus.

[0055] Other embodiments involve the polynucleotides of the inventioncontained in a variety of vectors. including plasmid, viral (e.g.,prokaryotic and eukaryotic viral vectors derived from Lambda phage,Herpesviruses, Adenovirus, Adeno-associated viruses, Cytomegalovirus,Vaccinia Virus, the M13-fl-fd family of viruses. retroviruses,Baculovirus, and others), phagemid, cosmid, and YAC (i.e., YeastArtificial Chromosome) vectors.

[0056] Yet other embodiments involve the polynucleotides of theinvention contained within heterologous polynucleotide environments.Polynucleotides of the invention have been inserted into heterologousgenomes, thereby creating transgenes, and transgenic animals, accordingto the invention. In particular, two types of gene fusions containingpartial murine cyr61 gene sequences have been used to generatetransgenic mice. (See below). One type of fused gene recombined thecoding sequence of cyr61 with one of three different promoters: 1) theK14 keratin promoter. 2) the β-actin promoter, or 3) thephosphoglycerokinase promoter. Adra et al. Gene 60:65-74 (1987). Thesefusion constructs were generated using standard techniques, as describedbelow in the context of a phosphoglycerokinase promoter (pgk-1)-cyr61fusion. An XhoI-ScaI genomic DNA fragment containing the entirecyr61coding region and all introns, but lacking the transcriptioninitiation site and polyadenylation signal. was cloned into plasmidpgk/β-gal, replacing the lacZ coding sequence. The resulting constructplaced cyr61 under the control of the strong pgk-1 promoter which isactive in all cells.

[0057] The second type of gene fusion recombined the cyr61 expressioncontrol sequences (i.e., promoter) with the E. coli β-galactosidasecoding sequence. The cyr61-lacZ fusion gene was constructed using thefollowing approach. A DNA fragment spanning nucleotides −2065 to +65relative to the transcription initiation nucleotide was used to replacethe pgk-1 promoter (Adra et al., Gene 60:65-74 [1987]) in plasmidpgk/β-gal by blunt end cloning. In addition, the polyadenylation signalfrom the bovine growth hormone gene was cloned into the plasmidcontaining the fusion gene. The resulting construct. Plasmid 2/lacZ. hasthe E. coli lacZ gene under the transcriptional control of a 2 kb DNAfragment containing the cyr61 promoter. The related plasmid 1.4/lacZ wasderived from plasmid 2lacZ by removing about 600 bp of cyr61 DNA foundupstream of an AflII site. Also, plasmid 2M/lacZ resembles Plasmid2/lacZ, except for a C-to-T transition in the CArG Box, created by PCR.These constructs were excised from the vectors by NotI digestion,purified using GeneClean [Bio 101, Inc., La Jolla, Calif.), and used togenerate transgenic mice (see below).

[0058] A cDNA fragment encoding mouse fisp12 has also been cloned usingstandard techniques. Ryseck et al., Cell Growth & Diff: 2:225-233(1991). incorporated herein by reference. The cloning was accomplishedby ligating an XhoII fragment containing the fisp12 cDNA coding regioninto Bat2HI-cleaved pBlueBacIII, a baculovirus expression vector(Invitrogen Corp., San Diego, Calif.). Recombinant baculovirus cloneswere obtained as described in Summers et al., TX Ag. Exp. Sta., Bulletin1555 (1987).

[0059] The human ortholog of fisp12, the gene encoding CTGF. was clonedby screening a fusion cDNA library with anti-Platelet-Derived GrowthFactor (anti-PDGF) antibodies, as described in U.S. Pat. No. 5,408,040.column 12, line 16. to column 13, line 29, incorporated herein byreference. The screening strategy exploited the immunologicalcross-reactivity of CTGF and PDGF.

[0060] The cloned copies of the Cyr61, fisp12, and ctgf cDNAs provide aready source for polynucleotide probes to facilitate the isolation ofgenomic coding regions, as well as allelic variants of the genomic DNAsor cDNAs. In addition, the existing cDNA clones, or clones isolated byprobing as described above, may be used to generate transgenicorganisms. For example, transgenic mice harboring cyr61 have beengenerated using standard techniques, as described in the next Example.

[0061] A clone, hCyr61 cDNA, containing the human cyr61 cDNA sequenceset forth in SEQ ID NO: 3, and a bacterial strain transformed with thatclone. Escherichia coli DH5α (hCyr61 cDNA), were deposited with theAmerican Type Culture Collection 12301 Parklawn Drive, Rockville, Md.20852 USA, on Mar. 14. 1997.

EXAMPLE 2 Sequence Analyses

[0062] The nucleotide sequence of murine cyr61 has been described,O'Brien et al. (1990); Latinikic et al., Nucl. Acids Res. 19:3261-3267(1991), and is set out herein as SEQ ID NO: 1.

[0063] The deduced amino acid sequence of murine Cyr61 has beenreported. O'Brien et al. (1990), and is set forth in SEQ ID NO: 2.

[0064] The nucleotide sequence of the human cyr61 cDNA was determinedusing the method of Sanger, as described in Sambrook et al. Sequencingtemplates were generated by constructing a series of nested deletionsfrom a pGEM-2 human cyr61 cDNA clone, as described in Example 1 above.The human cyr61 cDNA sequence is set forth in SEQ ID NO: 3. The aminoacid sequence of human Cyr61 was deduced from the human cyr61 cDNAsequence and is set forth in SEQ ID NO: 4.

[0065] A comparison of the mouse and human Cyr61 sequences. presented inSEQ ID NO: 2 and SEQ ID NO: 4, respectively, reveals 91% similarity.Both sequences exhibit an N-terminal signal sequence indicative of aprocessed and secreted protein; both proteins also contain 38 cysteineresidues. distributed throughout both proteins but notably absent fromthe central regions of both murine and human Cyr61. Notably, the regionof greatest sequence divergence between the mouse and human Cyr61 codingregions is this central region free of cysteine residues. However, the5′ untranslated regions of the mouse and human cyr61 cDNAs are even moredivergent (67% similarity). In contrast, the 3′ untranslated regions arethe most similar regions (91% similarity). In overall length, theencoded murine Cyr61 has 379 amino acids; human Cyr61 has 381 aminoacids.

[0066] A fisp12 cDNA sequence has also been determined and is set out inSEQ ID NO: 5. The amino acid sequence of Fisp12 has been deduced fromthe fisp12 cDNA sequence and is set forth in SEQ ID NO: 6. A comparisonof the amino acid sequences of murine Cyr61 and Fisp12 reveals that thetwo proteins are 65% identical. The structural similarity of Cyr61 andFisp12 is consistent with the similar functional properties of the twoproteins, described below.

[0067] A partial cDNA sequence of CTGF, containing the complete CTGFcoding region, has also been determined. The CTGF cDNA sequence wasobtained using M13 clones as templates for enzymatic sequencingreactions, as described. '040 Patent, at column 12, line 68 to column13, line 14 Additional cloning coupled with double-stranded enzymaticsequencing reactions, elucidated the entire sequence of the cDNAencoding CTGF. U.S. Pat. No. 5,408,040, column 14, line 44, to column15, line 8. incorporated herein by reference. The nucleotide sequence ofthe cDNA encoding CTGF is presented herein in SEQ ID NO: 7. The deducedamino acid sequence of the cDNA encoding CTGF is presented in SEQ ID NO:8.

EXAMPLE 3 RNA Analyses

[0068] Polynucleotide probes are useful diagnostic tools for angiogenic,and other, disorders correlated with Cyr61 expression because properlydesigned probes can reveal the location, and level, of cyr61 geneexpression at the transcriptional level. The expression of cyr61, inturn, indicates whether or not genes controlling the process ofangiogenesis are being expressed at typical. or expected, levels.

[0069] Using these tools. the mouse cyr61 mRNA expression pattern wasdetermined using an RNase protection technique. O'Brien et al., (1992).In particular. a 289 nucleotide antisense riboprobe was used that wouldprotect 246 nucleotides of the murine cyr61 mRNA (nucleotides 67 to 313using the numbering of O'Brien et al.) The assays showed levels of cyr61mRNA in PSA-1 cells (10 μg of total RNA) from either theundifferentiated state or stages 1, 2, and 3 of differentiation (PSA-1cells undergo three stages of cellular differentiation corresponding tomouse embryonic cells of the following gestational ages, in days:4.5-6.5 [PSA-1 stage 1]; 6.5-8.5 [PSA-1 stage 2]; 8.5-10.5 [PSA-1 stage3]). A comparison of the protection of whole embryonic and placentaltotal RNAs (20 μg each) showed that cyr61 is expressed in embryonictissues at times that are coincident with the processes of celldifferentiation and proliferation.

[0070] Expression characteristics of human cyr61 were determined byNorthern analyses. using techniques that are standard in the art.Sambrook et al. RNA was isolated from the human diploid fibroblasticcell line WI38 (ATCC CCL-75). In addition, RNA was isolated from ratcells (REF52). hamster cells (CHO), and monkey cells (BSC40). Each ofthe cell lines was grown to confluence in MEM-10 (Eagle's MinimalEssential Medium with Earle's salts [GIBCO-BRL. Inc.], 2 mM glutamine,and 10% fetal calf serum [fcs]) and maintained in MEM-0.5 (a 0.5% serummedium) for two days. Cultures were then stimulated with 20% fcs, in thepresence or absence of cycloheximide. by techniques known in the art.Lau et al. (1985: 1987). Ten microgram aliquots of RNA isolated fromthese cell lines were then fractionated by formaldehyde-agarose gelelectrophoresis, transferred and immobilized on nitrocellulose filters,and exposed to a full-length [³²p]-radiolabeled murine cyr61 cDNA probeunder hybridization conditions of high stringency. Human cyr61 RNAexpression was similar to murine cyr61 expression. Both mouse and humancyr61 expression yielded approximately 2 kilobase RNAs. Additionally.both mouse and human expression of Cyr61 were stimulated by serum andwere resistant to cycloheximide.

[0071] The distribution of human cyr61 mRNA was also examined usingmultiple tissue Northern blots (Clontech). The blots were hybridized inan ExpressHyb Solution (Clontech) according to the manufacturer'sinstructions. The results showed that cyr61 mRNA is abundant in thehuman heart. lung, pancreas. and placenta; is present at low levels inskeletal muscle, kidney and brain; and is not detectable in liver. Theseresults are consistent with the expression of cyr61 in mouse tissues.

[0072] In addition, total cellular RNA was isolated from human skinfibroblasts (HSFS) that were either quiescent, growing exponentially,stimulated by serum, or exposed to cycloheximide. HUVE cells (ATCC CRL1730) were maintained in Ham's F12 medium supplemented with 10% fbs(Intergene), 100 μg/ml heparin (Gibco BRL) and 30 μg/ml endothelial cellgrowth supplement (Collaborative Biomedical Products). Human skinfibroblasts (HSF, ATCC CRL-1475) and WI38 fibroblasts (ATCC CCL-75) weregrown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%fbs. Quiescent HSFs were prepared by growth in DMEM supplemented with10% fbs to confluence followed by changing the medium to DMEM containing0.1% fbs, for 2 days. Serum stimulation was carried out by changing themedium to 20% fibs for 1 hour. Where indicated. cycloheximide was addedto 10 μg/ml simultaneously with serum for 3 hours.

[0073] RNAs from the aforementioned cells were isolated using aguanidinium isothiocyanate protocol. Chonzcynski et al., Anal. Biochem.162:156-159 (1987). RNA samples were analyzed by electrophoreticseparation in formaldehyde-agarose gels followed by transfer to nylonfilters. Blots were hybridized with random-primed probes generated usingeither cyr61 or GAPDH as a template. Adams et al., Nature 355:632-634(1992). The results indicated that human cyr61 mRNA is not detectablypresent in quiescent human skin fibroblasts, is abundant inlogarithmically growing and serum stimulated HSFs. and is superinducedby cycloheximide.

[0074] The analysis of RNA encoding CTGF also involved techniques thatare standard in the art. In particular, investigation of RNA encodingCTGF involved the isolation of total cellular RNA and Northern analyses,performed as described in U.S. Pat. No. 5,408,040, column 11, line 59.to column 12. line 14. and column 13. lines 10-29, incorporated hereinby reference. A 2.4 kb RNA was identified. The expression of CTGF washigh in the placenta, lung, heart. kidney, skeletal muscle and pancreas.However, CTGF expression was low in the liver and brain.

EXAMPLE 4 Transgenic Animals

[0075] The construction of transgenic mice bearing integrated copies ofrecombinant cyr61 sequences was accomplished using linear DNA fragmentscontaining a fusion gene. The cyr61 coding sequence was independentlyfused to the β-actin, K14, and pgk promoters, described above.Expression of cyr61 was driven by these promoters in the transgenicanimals. The fusion gene was produced by appropriate restrictionendonuclease digestions, using standard techniques. The fusion genefragments were injected into single-cell zygotes of Swiss Webster mice.The injected zygotes were then implanted into pseudopregnant females.Several litters of mice were produced in this manner. Newbornsexhibiting unusual phenotypes were subjected to additional analyses. Forexample, neonatal transgenic mice expressing cyr61 under the pgkpromoter exhibited skeletal deformities. including curly tails. immobilejoints, and twisted limbs, resulting in locomotive difficulties. Thesemice typically were runted and died within seven days of birth.Transgenic mice expressing cyr61 under the β-actin promoter showed noobvious phenotype except that the mice were smaller. When mice bearingthe transgene were back-crossed to the in-bred strain C57 BL/6. theprogeny mice became progressively more runted with continuedback-crossing. After three to four such back-crosses, essentially noprogeny survive to reproduce. Transgenic mice expressing cyr61 under theK14 promoter exhibited a form of fibrotic dermatitis. The pathologyinvolved excessive surface scratching. sometimes resulting in bleeding.Transgenic organisms having knockout mutations of cyr61 can also becreated using these standard techniques. Hogan et al., Manipulating theMouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory Press1994), and are useful as models of disease states.

EXAMPLE 5 Cyr61 Expression

[0076] Native Cyr61 is expressed in embryonic tissues and is induced ina variety of wounded tissues. See below see also, O'Brien et al. (1992).The tissue distribution of Cyr61 was examined with rabbit anti-Cyr61polyclonal antibodies elicited using a conventional immunologicaltechnique (Harlow et al., 1987) and affinity-purified. Usingaffinity-purified anti-Cyr61 polyclonal antibodies according to theinvention, cyr61 expression was found in a variety of tissues, includingsmooth muscle, cardiomyocytes, and endothelia of the cardiovascularsystem; brain, spinal cord, ganglia and neurons. and retina of thenervous system; cartilage and bone of the skeletal system: epidermis,hair, oral epithelia, and cornea of the skin; bronchioles and bloodvessels of the lung: and placental tissues. In addition to expressionstudies directed towards native cyr61 (mRNA and protein), studies usingcyr61 transgenes, as described above, have contributed to ourunderstanding of Cyr61 expression. The use of transgene fusionscomprising the expression control sequences of cyr61 and the codingsequence of lacZ (encoding β-galactosidase) has provided a convenientcolorimetric assay for protein expression.

[0077] The colorimetric assay involves the use of5-Bromo-4-Chloro-3-Indolyl-β-D-Galactopyranoside (i. e., X-Gal) as asubstrate for β-galactosidase, the gene product of lacZ. Enzymaticcleavage of X-Gal by β-galactosidase produces an intensely coloredindigo dye useful in histochemical staining. In practice, embryonic andadult tissues subjected to analysis were dissected and fixed in 2%formaldehyde. 0.2% glutaraldehyde, 0.02% Nonidet P-40, and 0.01 sodiumdeoxycholate. in standard phosphate-buffered saline (PBS). Fixationtimes varied from 15-120 minutes, depending on the size and density oforgan or embryo samples being subjected to analysis. Subsequently,samples were rinsed in PBS and stained overnight at 37° C. in a PBSsolution containing 5 mM potassium ferrocyanide, 5 mM potassiumferricyanide, 2 mM MgCl₂, 0.02% Nonidet P-40, 0.01% sodium deoxycholateand 1 mg/ml of X-Gal (40 mg/ml in dimethylsulfoxide [DMSO]). Sampleswere then rinsed in PBS, post-fixed in 4% paraformaldehyde for 1-2hours. and stored in 70% ethanol at 4° C. until subjected to microscopicexamination. Mice containing the cyr61-lacZ transgene were used to mapthe expression profile of cyr61. The results are presented in Table Ifor embryonic tissues at day 12.5. TABLE I Transgenic Blood NervousMouse Line Vessels Skeleton System Epidermis 1.S¹ +² − + + 2.S + + + +3.S + +/− + + 4.T + − − NA 5.T + − − NA 6.T + +/− − NA 7.T + +/− − NA8.T + +/− + NA

[0078] The results indicate that Cyr61 is expressed in a variety ofembryonic cell types. Additional information has been gleaned from theectopic expression of Cyr61 resulting from another type of transgenefusion comprising a heterologous expression control sequence coupled tothe coding sequence of cyr61. The control sequences, the K14 keratinpromoter, the β-actin promoter, and the phosphoglycerokinase promoter,directed the expression of Cyr61 in a pattern that differed from itsnative expression.

[0079] Transgenic mice ectopically expressing Cyr61 were routinelysmaller than wild type mice and exhibited a reduction in average lifespan. Moreover, these transgenic mice had abnormal hearts (i.e.,thickened chamber walls with a corresponding reduction in internalcapacity) and abnormal skeletons characterized by curved spines, jointsswollen to the point of immobility. and curly tails. Therefore, ectopicexpression of Cyr61 interferes with angiogenesis (blood vesseldevelopment and heart development) and chondrogenesis (skeletaldevelopment). In addition. transgenic mice carrying knockout mutationsof cyr61 may be developed and tested as models of disease statesassociated with a lack of Cyr61 activity.

[0080] A strategy for the expression of recombinant cyr61 was designedusing a Baculovirus expression vector in Sf9 cells. Expression systemsinvolving Baculovirus expression vectors and Sf9 cells are described inCurrent Protocols in Molecular Biology §§ 16.9.1-16.12.6 (Ausubel et al.eds. 1987). One embodiment of the present invention implemented theexpression strategy by cloning the murine cyr61 cDNA into pBlueBac2 atransfer vector. The recombinant clone, along with target AcMNPV (i.e.,Autographa californica nuclear polyhedrosis virus, or Baculovirus) DNA,were delivered into Sf9 cells by liposome-mediated transfection, usingthe MaxBac Kit (Invitrogen, Inc., San Diego, Calif.) according to themanufacturer's instructions. Recombinant virus was plaque-purified andamplified by 3 passages through Sf9 cells via infection.

[0081] Conditioned medium of Sf9 insect cells infected with abaculovirus construct driving the synthesis of murine Cyr61 was used asa source for purification of Cyr61 (see below). The purified recombinantCyr61 retains certain characteristics of the endogenous protein, e.g.,the heparin-binding activity of Cyr61 (described below) from 3T3fibroblast cells and had a structure similar to the endogenous proteinas revealed by independent peptide profiles produced by partialproteolysis using either chymotrypsin or trypsin (sequencing grade:Boehringer-Mannheim. Inc. Indianapolis. Ind.).

[0082] Human cyr61 was also expressed using the baculovirus system. ASmaI-HindIII fragment (corresponding to nucleotides 100-1649 of SEQ IDNO: 3) of cyr61 cDNA spanning the entire human cyr61 open reading framewas subcloned into a pBlueBac3 baculovirus expression vector(Invitrogen). Recombinant baculovirus clones were obtained, plaquepurified and amplified through three passages of Sf9 infection, usingconventional techniques. Infection of Sf9 cells and human Cyr61 (hCyr61)purification was performed using standard techniques, with somemodifications. Sf9 cells were maintained in serum-free Sf900-II medium(Sigma). Sf9 cells were seeded, at 2-3×10⁶ cells per 150 mm dish, inmonolayer cultures and were infected with 5 plaque forming units (PFU)of recombinant virus per cell. The conditioned medium was collected at 8and 96 hours post-infection. cleared by centrifugation (5000×g. 5minutes) and adjusted to 50 mM MES [2-(N-Morpholino)ethanesulfonicacid], pH 6.0, 1 mM PMSF (phenylmethylsulfonyl fluoride), and 1 mM EDTA.The medium was mixed with Sepharose S beads equilibrated with loadingbuffer (50 mM MES, pH 6.0, 1 mM PMSF, 1 mM EDTA. 150 mM NaCl) at a ratioof 5 ml Sepharose S beads per 500 ml of conditioned medium and theproteins were allowed to bind to the Sepharose S at 4° C. (o/n) withgentle stirring. Sepharose S beads were collected by sedimentationwithout stirring for 20 minutes and applied to the column. The columnwas washed with 6 volumes of 0.3 M NaCl in loading buffer andrecombinant human Cyr61 was eluted from the column with a step gradientof NaCl (0.4-0.8 M) in loading buffer. This procedure resulted in 3-4milligrams of purified Cyr61 protein from 500 ml of conditioned medium,and the purified Cyr61 was over 90% pure as judged by Coomassie Bluestaining of SDS-gels.

[0083] In another embodiment, the complete human cyr61 cDNA is clonedinto a cytomegalovirus vector such as pBK-CMV (Stratagene, La Jolla,Calif.) using the Polymerase Chain Reaction (Hayashi, in PCR: ThePolymerase Chain Reaction 3-13 [Mullis et al. eds., Birkhauser 1994])and Taq Polymerase with editing function, followed by conventionalcloning techniques to insert the PCR fragment into a vector. Theexpression vector is then introduced into HUVE cells byliposome-mediated transfection. Recipient clones containing thevector-borne neo gene are selected using G418. Selected clones areexpanded and Cyr61 expression is identified by ReverseTranscription-Polymerase Chain Reaction (i.e., RT-PCR; Chelly et al, inPCR: The Polymerase Chain Reaction 97-109 [Mullis et al. eds.,Birkhauser 1994]) or Enzyme-Linked Immunosorbent Assays (i.e., ELISA;Stites et al., in Basic and Clinical Immunology 243 [Stites et al. eds.,Appleton & Lange 1991]) assays.

[0084] In other embodiments of the invention, Cyr61 protein is expressedin bacterial cells or other expression systems (e.g., yeast) using, thecyr61 cDNA coding region linked to promoters that are operative in thecell type being used. Using one of these approaches, Cyr61 protein maybe obtained in a form that can be administered directly to patients,e.g., by intravenous routes, to treat angiogenic, chondrogenic, oroncogenic disorders. One of skill in the art would recognize that otheradministration routes are also available. e.g., topical or localapplication, liposome-mediated delivery techniques, or subcutaneous,intradermal, intraperitoneal, or intramuscular injection.

EXAMPLE 6 Fisp12 Expression

[0085] The expression of Fisp12, and a comparison of the expressioncharacteristics of Cyr61 and Fisp12, were investigated usingimmunohistochemical techniques. For these immunohistochemical analyses.tissue samples (see below) were initially subjected to methyl-Carnoy'sfixative (60% methanol. 30% chloroform and 10% glacial acetic acid) for2-4 hours. They were then dehydrated, cleared and infiltrated inParaplast X-tra wax at 55-56 C for minimal duration. 7 μm thick sectionswere collected on poly-L-lysine-coated slides (Sigma), mounted anddewaxed. They were then treated with 0.03% solution of H₂O₂ in methanolfor 30 min. to inactivate endogenous peroxidase activity. Afterrehydration. sections were put in Tris-buffered saline (TBS: 10 mM Tris.pH 7.6 and 140 mM NaCl) for 15 minutes. At that point, sections wereblotted to remove excess TBS with paper towels and blocked with 3%normal goat serum in TBS for 10 minutes in a humid chamber. Excessbuffer was then drained and primary antibodies applied. Affinitypurified anti-Cyr61 antibodies were diluted 1:50 in 3% normal goatserum-TBS solution. Dilution for affinity-purified anti-Fisp12 antibodywas 1:25. Routine control was 3% normal goat serum-TBS, or irrelevantantibody (for example, monoclonal anti-smooth muscle cell α-actin).Specificity of staining was confirmed by incubation of anti-Cyr61 oranti-Fisp12 antibodies with an excess of the corresponding antigen onice for at least two hours prior to applying to sections. Completecompetition was observed. By contrast, cross-competition (incubation ofanti-Cyr61 antibodies with Fisp12 antigen and vice versa) did not occur.

[0086] Primary antibodies were left on sections overnight at 4 C. Theywere then washed with TBS twice, and subjected to 30 minutes incubationwith secondary antibodies at room temperature. Secondary antibodies usedwere goat anti-rabbit horseradish peroxidase conjugates fromBoehringer-Mannheim, Inc., Indianapolis, Ind. (used at 1:400 dilution).Sections were washed twice in TBS and chromogenic horseradish peroxidasesubstrate was applied for 5 minutes (1 mg/ml of diaminobenzidine in 50mM Tris-HCl. pH 7.2 and 0.03% H₂O₂). Sections were then counterstainedin Ehrlich's haematoxylin or in Alcian blue, dehydrated and mounted inPermount.

[0087] Mouse embryos between the neural fold (E8.5) and lateorganogenesis (E18.5) stages of development were sectioned and subjectedto immunostaining with antigen-affinity-purified rabbit anti-Cyr61 andanti-Fisp12 antibodies. As various organs developed duringembryogenesis. the presence of Cyr61 and Fisp12 was determined. Cyr61and Fisp12 were co-localized in a number of tissues and organs. Anotable example is the placenta, where both proteins were readilydetectable. In particular. both Cyr61 and Fisp12 were found in andaround the trophoblastic giant cells. corroborating the previousdetection of cyr61 mRNA in these cells by in situ hybridization (O'Brienand Lau, 1992). Both Cyr61 and Fisp12 signals in immunohistochemicalstaining were blocked by either the corresponding Cyr61 or Fisp 12antigen but not by each other, nor by irrelevant proteins, demonstratingspecificity. In general, Cyr61 and Fisp12 proteins could be detectedboth intracellularly and extracellularly.

[0088] In addition to the placenta, both Cyr61 and Fisp12 were detectedin the cardiovascular system, including the smooth muscle, thecardiomyocytes, and the endothelia. Both proteins were also found in thebronchioles and the blood vessels in the lung. Low levels of anti-Cyr61and anti-Fisp12 staining could be detected transiently in the skeletalmuscle. This staining is associated with connective tissue sheets,rather than myocytes; in this instance the staining pattern was clearlyextracellular.

[0089] A more complex pattern of distribution was found in the epidermisand the epithelia. Both Cyr61 and Fisp12 staining could be detected inthe early, single-cell layer of embryonic epidermis, as well as inlater, multilayered differentiating epidermis. Fisp12 in epidermisdeclined to an undetectable level by the end of gestation and remainedas such through adulthood, whereas Cyr61 was readily detectable in theepidermis. In the neonate, a strong staining for Fisp12 was seen in theoral epithelia where Cyr61 staining was much weaker, while Cyr61 wasfound in the upper jawbone where Fisp12 was not observed. Theanti-Fisp12 signal in the oral epithelia gradually increased andremained intense into adulthood. In the tongue, both Cyr61 and Fisp12were seen in the keratinized epithelia, although the Fisp12 stainingpattern, but not that of Cyr61, excludes the filiform papillae.

[0090] Aside from the aforementioned sites of localization, Cyr61 andFisp12 were also uniquely localized in several organ systems. Forexample, Cyr61. but not Fisp12, was present in skeletal and nervoussystems. As expected from in situ hybridization results (O'Brien andLau, 1992), Cyr61 protein was readily detected in the sclerotomal massesof the somites, and in cartilage and bone at later stages ofdevelopment. In contrast, Fisp12 was not detectable in the skeletalsystem. Since correlation with chondrocytic differentiation is one ofthe most striking features of cyr61 expression (O'Brien and Lau, 1992),the absence of Fisp12 in the skeletal system may underscore an importantdifference in the biological roles of Cyr61 and Fisp12. In the E14.5embryo, Cyr61 could be detected in the ventral spinal cord, dorsalganglia, axial muscle and sclerotome-derived cartilaginous vertebrae.Fisp12, however, was not detected in these tissues.

[0091] By contrast, Fisp12 was uniquely present in various secretorytissues. Beginning at E16.5. Fisp12 could be detected in the pancreas,kidneys, and salivary glands. In the pancreas, Fisp12 was strictlylocalized to the periphery of the islets of Langerhans. In the kidney,strong Fisp12 staining was seen in the collecting tubules and Henle'sloops, regions where Cyr61 was not found. In the mucous-typesubmandibular salivary gland only collecting ducts stained for Fisp12,whereas in the mixed mucous-serous submandibular gland, both serousacini and collecting ducts stained. The signal in acini was peripheral,raising the possibility that Fisp12 is capsule-associated. In simpleholocrine sebaceous glands a strong acellular Fisp 12 signal wasdetected.

[0092] In summary, Cyr61 and Fisp12 have been co-localized in theplacenta. the cardiovascular system, the lung and the skin. Neitherprotein was detected in the digestive system or the endocrine glands.Unique localization of Cyr61 can be detected in the skeletal and centralnervous system. and Fisp12 is found in secretory tissues where Cyr61 isnot.

[0093] An issue closely related to protein expression concerns themetabolic fate of the expressed proteins. Members of the cysteine-richprotein family have been localized. As discussed above, secreted Cyr61is found in the ECM and on the cell surface but not in the culturemedium (Yang and Lau, 1991), yet secreted Fisp12 was readily detected inthe culture medium (Ryseck et al., 1991). To address the question ofwhether Fisp12 is also ECM-associated. the fate of both Cyr61 and Fisp12was followed using pulse-chase experiments. Serum-stimulated.sub-confluent NIH 3T3 fibroblasts were metabolically pulse-labeled for 1hour and chased in cold medium for various times. Samples werefractionated into cellular, ECM, and medium fractions followed byimmunoprecipitation to detect Cyr61 and Fisp12. Both proteins have asimilar short half-life of approximately 30 minutes in the cellularfraction, which includes both newly synthesized intracellular proteinsas well as secreted proteins associated with the cell surface (Yang andLau, 1991). It should be noted that since Cyr61 is quantitativelysecreted after synthesis and only a minor fraction is stably associatedwith the ECM, the bulk of secreted Cyr61 is cell-surface associated(Yang and Lau, 1991).

[0094] A fraction of Cyr61 was chased into the ECM where it remainedstable for several hours. Newly synthesized Fisp12 was also chased intothe ECM, where its half-life was only about 1 hour. A larger fraction ofFisp12 was chased to the conditioned medium, where no Cyr61 wasdetectable. Fisp12 in the conditioned medium also had a short half-lifeof about 2 hours. Thus, whereas Cyr61 is strongly associated with theECM, Fisp12 is associated with the ECM more transiently. This resultsuggests that Fisp12 might be able to act at a site distant from itssite of synthesis and secretion. whereas Cyr61 may act more locally.

[0095] Since many ECM proteins associate with the matrix via interactionwith heparan sulfate proteoglycans, the affinity with which a proteinbinds heparin might be a factor in its interaction with the ECM. Theresults of heparin binding assays, described below, are consistent withthis hypothesis.

EXAMPLE 7 Protein Purification

[0096] Serum-stimulated NIH 3T3 fibroblast cells were lysed to provide asource of native murine Cyr61. Yang et al. Similarly, human fibroblastsare a source of native human Cyr61.

[0097] Recombinant murine Cyr61 was purified from Sf9 cells harboringthe recombinant Baculovirus vector, described above, containing thecomplete cyr61 coding sequence. Although murine Cyr61 in Sf9 celllysates formed insoluble aggregates as was the case with bacterial cellextracts, approximately 10% of the Cyr61 synthesized was secreted intothe medium in a soluble form. The soluble, secreted form of Cyr61 wastherefore subjected to purification.

[0098] Initially. subconfluent Sf9 cells in monolayer cultures weregenerated in supplemented Grace's medium (GIBCO-BRL, Inc., Grand Island,N.Y.). Grace, Nature 195:788 (1962). The Sf9 cells were then infectedwith 10 plaque-forming-units/cell of the recombinant Baculovirus vector,incubated for 16 hours, and fed with serum-free Grace's medium. Thesecells were expanded in serum-free Grace's Medium. The conditioned mediumwas collected 48 hours post-infection, although Cyr61 expression couldbe detected in the medium 24 hours after infection. Subsequently, theconditioned medium was cleared by centrifugation at 5000×g for 5minutes, chilled to 4° C., adjusted to 50 mM MES, pH 6.0, 2 mM EDTA(Ethylenediamine tetraacetic acid), 1 mM PMSF (Phenylmethylsulfonylfluoride) and applied to a Sepharose S column (Sigma Chemical Co., St.Louis, Mo.) at 4° C. (5 ml void volume per 500 ml medium). The columnwas washed with a buffer (50 mM MES, pH 6.0, 2 mM EDTA, 0.5 mM PMSF)containing 150 NaCl. and bound proteins were eluted with a lineargradient of NaCl (0.2-1.0 M) in the same buffer. The pooled fractions ofCyr61 eluted at 0.6-0.7 M NaCl as a distinct broad peak. The columnfractions were 90% pure, as determined by 10% SDS-PAGE followed byCoomassie Blue staining or Western analysis. using techniques that arestandard in the art. Yang et al.; see also, Sambrook et al., supra. ForWestern analysis, blots were probed with affinity-purified anti-Cyr61antibodies as described in Yang et al., supra. After antibody probing,Western blots were stained with ECL™ (i.e., Enhanced ChemiLuminescence)detection reagents (Amersham Corp., Arlington Heights. Ill.). Fractionscontaining Cyr61 were pooled, adjusted to pH 7.5 with Tris-HCl. pH 7.5,and glycerol was added to 10% prior to storage of the aliquots at −70°C. Protein concentration was determined by the modified Lowry methodusing the BioRad protein assay kit (BioRad Laboratories, Inc., Hercules.Calif.). This purification procedure was repeated at least five timeswith similar results. The typical yield was 3-4 mg of 90% pure Cyr61protein from 500 ml of conditioned medium.

[0099] Fisp12 was purified using a modification of the Cyr61purification scheme (Kireeva et al., Experimental Cell Research, inpress). Serum-free conditioned media (500 ml) of Sf9 cells infected at10 pfu per cell were collected 48 hours post-infection and loaded onto a5-ml Sepharose S (Sigma Chemical Co., St. Louis, Mo.) column. Afterextensive washing at 0.2 M and 0.4 M NaCl. bound proteins were recoveredby step elution with 50 mM MES (pH 6.0) containing 0.5 M NaCl. Fractionscontaining Fisp12 of greater than 80% purity were pooled, NaCl adjustedto 0.15 M and the protein was concentrated 3-5 fold on a 0.5 mlSepharose S column with elution of the protein at 0.6 M NaCl.

[0100] This purification scheme allowed the isolation of 1.5 mg ofrecombinant Fisp12 protein of at least 80% purity from 500 ml ofserum-free conditioned media.

[0101] CTGF was purified by affinity chromatography using anti-PDGFcross-reactivity between CTGF and PDGF, as described in U.S. Pat. No.5.408.040. column 7, line 15, to column 9, line 63, incorporated hereinby reference.

EXAMPLE 8 Polypeptide Characterization

[0102] The murine Cyr61 protein has a M of 41,000 and is 379 amino acidslong including the N-terminal secretory signal. There is 91% amino acidsequence identity with the 381 amino acid sequence of the human protein.Those regions of the mouse and human proteins contributing to thesimilarity of the two proteins would be expected to participate in thebiological activities shared by the two polypeptides and disclosedherein. However, the mouse and human proteins do diverge significantlyin the central portion of the proteins, where each protein is devoid ofcysteines. See, O'Brien et al., Cell Growth & Diff 3:645-654 (1992). Acysteine-free region in the murine Cyr61 amino acid sequence is foundbetween amino acid residues 164 to 226 (SEQ ID NO: 2). A correspondingcysteine-free region is found in the human Cyr61 amino acid sequencebetween amino acid residues 163 to 229 (SEQ ID NO: 4). Moreparticularly, the mouse and human Cyr61 proteins are most divergentbetween Cyr61 amino acids 170-185 and 210-225. Other members of the ECMsignalling molecule family of cysteine-rich proteins, e.g., Fisp12 (SEQID NO: 6) and CTGF (SEQ ID NO: 8), exhibit similar structures suggestiveof secreted proteins having sequences dominated by cysteine residues.

[0103] Because murine Cyr61 contains 38 cysteines in the 355 amino acidsecreted portion, the contribution of disulfide bond formation to Cyr61tertiary structure was investigated. Exposure of Cyr61 to 10 mMdithiothreitol (DTT) for 16 hours did not affect the ability of Cyr61 tomediate cell attachment (see below). However, Cyr61 was inactivated byheating at 75° C. for 5 minutes by incubation in 100 mM HCl, or uponextensive digestion with chymotrypsin. These results indicate thatmurine Cyr61 is a heat- and acid-labile protein whose activeconformation is not sensitive to reducing agents. The aforementionedstructural similarities of murine and human Cyr61 polypeptides suggeststhat human Cyr61 may also be sensitive to heat or acid, but insensitiveto reducing agents. In addition, Cyr61 is neither phosphorylated norglycosylated.

[0104] To determine if the purified recombinant murine Cyr61 describedabove was the same as native murine Cyr61, two additionalcharacteristics of mouse Cyr61 were determined. First, two independentprotein fingerprints of recombinant and native murine Cyr61 wereobtained. Purified recombinant murine Cyr61 and a lysate ofserum-stimulated 3T3 cells. known to contain native murine Cyr61, weresubjected to limited proteolysis with either trypsin or chymotrypsin,and their digestion products were compared. Partial tryptic digests ofboth the recombinant protein and cell lysate resulted in two Cyr61fragments of approximately 21 and 19 kDa. Similarly, fingerprinting ofboth preparations by partial chymotrypsin digestion produced stable 23kDa fragments from recombinant murine Cyr61 and native murine Cyr61.

[0105] Another criterion used to assess the properties of recombinantCyr61 was its ability to bind heparin, described below. Purifiedrecombinant murine Cyr61 bound quantitatively to heparin-sepharose at0.15 M NaCl and was eluted at 0.8-1.0 M NaCl. This heparin bindingcapacity is similar to native murine Cyr61 obtained fromserum-stimulated mouse fibroblasts. Because of the similarities of themurine and human Cyr61 proteins, recombinant human Cyr61 should exhibitproperties similar to the native human Cyr61, as was the case for themurine polypeptides.

[0106] The polypeptides of the invention also extend to fragments.analogs and derivatives of the aforementioned full-length ECMsignalling-molecules such as human and mouse Cyr61. The inventioncontemplates peptide fragments of ECM signalling molecules that retainat least one biological activity of an ECM signalling molecule, asdescribed above. Candidate fragments for retaining at least onebiological activity of an ECM signalling molecule include fragments thathave an amino acid sequence corresponding to a conserved region of theknown ECM signalling molecules. For example, fragments retaining one ormore of the conserved cysteine residues of ECM signalling moleculeswould be likely candidates for ECM signalling molecule fragments thatretain at least one biological activity. Beyond the naturally occurringamino acid sequences of ECM signalling molecule fragments, thepolypeptides of the invention include analogs of the amino acidsequences or subsequences of native ECM signalling molecules.

[0107] ECM signalling molecule analogs are polypeptides that differ inamino acid sequence from native ECM signalling molecules but retain atleast one biological activity of a native ECM signalling molecule, asdescribed above. These analogs may differ in amino acid sequence fromnative ECM signalling molecules, e.g., by the insertion, deletion, orconservative substitution of amino acids. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes can be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyte etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge, andinclude the following values: alanine (+1.8), arginine (−4.5),asparagine (−3.5), aspartate (−3.5), cysteine/cystine (+2.5), glycine(−0.4), glutamate (−3.5), glutamine (−3.5). histidine (−3.2). isoleucine(+4.5), leucine (+3.8), lysine (−3.9), methionine (+1.9). phenylalanine(+2.8), proline (−1.6), serine (−0.8), threonine (−0.7), tryptophan(−0.9). tyrosine (−1.3), and valine (+4.2). It is known in the art thatamino acids of similar hydropathic indexes can be substituted and stillretain protein function. Preferably, amino acids having hydropathicindexes of ±2 are substituted.

[0108] The hydrophilicity of amino acids can also be used to revealsubstitutions that would result in proteins retaining biologicalfunction. A consideration of the hydrophilicity of amino acids in thecontext of a polypeptide permits calculation of the greatest localaverage hydrophilicity of that polypeptide, a useful measure that hasbeen reported to correlate well with antigenicity and immunogenicity.U.S. Pat. No. 4,554,101. incorporated herein by reference.Hydrophilicity values for each of the common amino acids. as reported inU.S. Pat. No. 4,554,101, are: alanine (−0.5), arginine (+3.0).asparagine (+0.2), aspartate (+3.0±1), cysteine (−1.0). glycine (0),glutamate (+3.0±1), glutamine (+0.2). histidine (−0.5), isoleucine(−1.8), leucine (−1.8), lysine (+3.0), methionine (−1.3), phenylalanine(−2.5), proline (−0.5±1), serine (+0.3), threonine (−0.4), tryptophan(−3.4). tyrosine (−2.3). and valine (−1.5). Substitution of amino acidshaving similar hydrophilicity values can result in proteins retainingbiological activity. for example immunogenicity, as is understood in theart. Preferably, substitutions are performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hyrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

[0109] Additionally, computerized algorithms are available to assist inpredicting amino acid sequence domains likely to be accessible to anaqueous solvent. These domains are known in the art to frequently bedisposed towards the exterior of a protein, thereby potentiallycontributing to binding determinants, including antigenic determinants.Having the DNA sequence in hand, the preparation of such analogs isaccomplished by methods well known in the art (e.g., site-directed)mutagenesis and other techniques.

[0110] Derivatives of ECM signalling molecules are also contemplated bythe invention. ECM signalling molecule derivatives are proteins orpeptides that differ from native ECM signalling molecules in ways otherthan primary structure (i.e., amino acid sequence). By way ofillustration, ECM signalling molecule derivatives may differ from nativeECM signalling molecules by being glycosylated, one form ofpost-translational modification. For example, polypeptides may exhibitglycosylation patterns due to expression in heterologous systems. Ifthese polypeptides retain at least one biological activity of a nativeECM signalling molecule, then these polypeptides are ECM signallingmolecule derivatives according to the invention. Other ECM signallingmolecule derivatives include, but are not limited to, fusion proteinshaving a covalently modified N- or C-terminus, PEGylated polypeptides,polypeptides associated with lipid moieties, alkylated polypeptides.polypeptides linked via an amino acid side-chain functional group toother polypeptides or chemicals and additional modifications as would beunderstood in the art. In addition. the invention contemplates ECMsignalling molecule-related polypeptides that bind to an ECM signallingmolecule receptor, as described below.

[0111] The various polypeptides of the present invention, as describedabove may be provided as discrete polypeptides or be linked, e.g., bycovalent bonds, to other compounds. For example, immunogenic carrierssuch as Keyhole Limpet Hemocyanin may be bound to a ECM signallingmolecule of the invention.

EXAMPLE 9 Heparin Binding Assay

[0112] The heparin binding assay for native murine Cyr61. described inYang et al., was modified for the purified recombinant murine protein.Initially, recombinant purified Cyr61 was suspended in RIPA(Radioimmuno-precipitation assay) buffer (150 mM NaCl, 1.0% NP-40, 0.5%deoxycholate. 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 1 mMphenylmethylsulfonyl fluoride). Next. 200 μl of a 50% (v/v) slurry ofheparin-Sepharose CL 6 B beads (Pharmacia-LKB Biotechnology, Inc.,Piscataway, N.J.) was added to 100 μl of the recombinant Cyr61 solutionand incubated for 1 hour. Under these conditions, human Cyr61 wasquantitatively bound to heparin-agarose. Application of a saltconcentration gradient in RIPA buffer resulted in the elution ofrecombinant murine Cyr61 at 0.8-1.0 M NaCl. The elution profile of therecombinant protein was similar to the elution profile for native murineCyr61.

[0113] One might expect that Fisp12 would bind heparin with loweraffinity than Cyr61, as it does not interact with the ECM as strongly asCyr61. To examine this possibility. metabolically labeled [³⁵S-cysteine;100 μCi per 100 mm dish: ICN] cell lysates were incubated with heparinagarose beads which were subsequently washed to remove unbound proteins.Bound proteins were eluted in increasing salt concentrations. Fisp12from cell lysates was retained on heparin agarose but was eluted by 0.2to 0.6 M NaCl with peak elution at 0.4 M NaCl. This is in contrast toCyr61, which was eluted at significantly higher concentrations of NaCl.This difference in heparin binding is consistent with the differingaffinities of Cyr61 and Fisp12 for the ECM. suggesting that binding toheparan sulfate proteoglycans may be a primary mechanism by which bothproteins associate with the ECM.

EXAMPLE 10 Receptors

[0114] Human Cyr61, like murine Cyr61, was localized to the cell surfaceand ECM. The localization of Cyr61 to the cell surface implicated a cellsurface receptor binding Cyr61. Consistent with that implication. thebiological effects of Cyr61 are mediated by the α_(v)β₃ integrin, orvitronectin receptor. The α_(v)β₃ integrin, in association with otherintegrins, forms protein clusters providing focal points forcytoskeletal attachment. Cyr61 induces the formation of proteinclusters, including the protein clusters containing the α_(v)β₃integrin. In addition, using an in vitro assay, the biological effectsof Cyr61, including Cyr61-induced cell adhesion and mitogenesis, wereabolished by the addition of either one of two monoclonal antibodies-LM609 (Cheresh, Proc. Natl. Acad. Sci. [USA] 84:6471-6475 [1987]) oranti-VnR 1 (Chen et al., Blood 86:2606-2615 [1995])—directed to theα_(v)β₃ integrin. This data led to the identification of the α_(v)β₃integrin as the Cyr61 receptor.

[0115] Cyr61 induction of HUVE cell adhesion, described in Example 13below, led to an investigation of the divalent cation-sensitive cellsurface receptors expressed by HUVE cells. The cell adhesion propertiesof Cyr61 were used to identify the receptor, which is a divalentcation-sensitive cell surface receptor. The ability of Cyr61 to mediatecell adhesion, coupled with the strict requirement for divalent cationsin the process, indicated that Cyr61 interacts with one of the divalentcation-dependent cell adhesion molecules from the integrin, selectin, orcadherin families. Ruoslahti et al., Exp. Cell Res. 227:1-11 (1996).Using well-characterized approaches to receptor identification, a seriesof inhibition studies were conducted. Inhibitors, or blocking agents, ofvarious degrees of specificity (EDTA, similar to the EGTA describedabove; inhibitory peptides bearing variants of the RGD (single letteramino acid code) integrin recognition motif, such as RGDS, SGDR, andRGDSPK (Ruoslahti, et al., Science 238:491-497 [1987], Ruoslahti, E.,Ann. Rev. of Cell and Dev. Biol. 12:698-715 [1996]); and known. specificanti-receptor antibodies) were used to identify a Cyr61 receptor. Thatreceptor was the α_(v)β₃ integrin, also known to function as thevitronectin receptor. Confirmation of that identification was obtainedby showing that antibody LM609, a specific anti-α_(v)β₃ integrinantibody. could block the effect of Cyr61 on cell adhesion. Integrinsform a large family of heterodimeric adhesion receptors, with a broadligand specificity range, involved in cell-cell and cell-matrixinteractions. Beyond their requirement for divalent cations and theirinvolvement in cell-matrix adhesion events [Hynes, R. O., Cell 69:11-25(1992)], integrins also are involved in cell migration [Damsky et al.,Curr. Opin. Cell Biol. 4:772-781 (1992): Doerr et al., J. Biol. Chem.271:2443-447 (1996)] and proliferation [Juliano et al., J. Cell Biol.,120:577-585 (1993); Plopper et al., Mol. Biol. Cell 6:1349-1365 (1995);and Clark et al., Science 268:233-239 (1995)], two additional processesassociated with Cyr61 activity. The α_(v)β₃ integrin was found to beessential for Cyr61-mediated cell adhesion.

[0116] Characterization of CTGF binding to cells has been reported tooccur through a cell surface receptor that also interacts with PDGF-BB(the BB isoform of PDGF), as recited in U.S. Pat. No. 5,408,040, column11, line 10. to column 12, line 14, incorporated herein by reference.The identification of the foregoing receptors permits the the design andproduction of molecules and which bind to the respective receptors toinhibit the activities of ECM molecules.

EXAMPLE 11 Anti-ECM Signalling Molecule Antibodies

[0117] Antibodies, optionally attached to a label or to a toxin asdescribed below, are also contemplated by the present invention. Theavailability of the human cyr61 cDNA sequence and the Cyr61 deducedprotein sequence facilitate the implementation of methods designed toelicit anti-Cyr61 antibodies using a number of techniques that arestandard in the art. Harlow et al.

[0118] In one embodiment, polyclonal antibodies directed against Cyr61are generated. The generation of anti-Cyr61 antibodies specific forhuman Cyr61, for example, is optimized by designing appropriateantigens. The human Cyr61 protein is 381 amino acids long, including theN-terminal secretory signal. As described above, human Cyr61 exhibits a91% amino acid sequence identity with the 379 amino acid sequence of themouse protein. However. the mouse and human proteins diverge mostsignificantly in the central portion of the proteins, where they aredevoid of cysteines (see above). These sequence differences areexploited to elicit antibodies specific to the human Cyr61 by using asan antigen a peptide having a sequence derived from one of the divergentregions in the human protein, although antibodies directed to aconserved region are also contemplated by the invention.

[0119] In another embodiment of the present invention, monoclonalantibodies are elicited using intact recombinant human Cyr61 although afragment may be used. Female BALB/c mice are inoculatedintraperitoneally with a mixture of 0.25 ml recombinant human Cyr61(5-50 micrograms), bacterially produced or produced in eukaryotic cells,and 0.25 ml complete Freund's adjuvant. Fourteen days later theinjections are repeated with the substitution of incomplete Freund'sadjuvant for complete Freund's adjuvant. After an additional two weeks,another injection of human Cyr61 in incomplete Freund's adjuvant isadministered. About two weeks after the third injection, tail bleeds areperformed and serum samples are screened for human anti-Cyr61 antibodiesby immunoprecipitation with radiolabeled recombinant human Cyr61. Abouttwo months after the initial injection, mice whose sera yield thehighest antibody titers are given booster injections of Cyr61 (5-50micrograms in incomplete Freund's adjuvant, 0.1 ml intravenously and 0.1ml intraperitoneally). Three days after the booster injection, the miceare sacrificed. Splenocytes are then isolated from each mouse usingstandard techniques, and the cells are washed and individually fusedwith a myeloma cell line, e.g., the X63Ag8.653 cell line (Harlow etal.), using polyethylene glycol, by techniques that are known in theart. Other suitable cell lines for fusion with splenocytes are describedin Harlow et al., at page 144, Table 6.2, incorporated herein byreference. Fused cells are removed from the PEG solution, diluted into acounter-selective medium (e.g., Hypoxanthine-Aminopterin-Thymidine orHAT medium) to kill unfused myeloma cells. and inoculated intomulti-well tissue culture dishes.

[0120] About 1-2 weeks later, samples of the tissue culture supernatantsare removed from wells containing growing hybridomas, and tested for thepresence of anti-Cyr61 antibodies by binding to recombinant human Cyr61bound to nitrocellulose and screening with labeled anti-immunoglobulinantibody in a standard antibody-capture assay. Cells from positive wellsare grown and single cells are cloned on feeder layers of splenocytes.The cloned cell lines are stored frozen. Monoclonal antibodies arecollected and purified using standard techniques, e.g., hydroxylapatitechromatography. In an alternative, Cyr61 peptides used as antigens. maybe attached to immunogenic carriers such as keyhole limpet hemocyanincarrier protein. to elicit monoclonal anti-Cyr61 antibodies.

[0121] Another embodiment involves the generation of antibody productsagainst a fusion protein containing part, or all, of human Cyr61,including enough of the protein sequence to exhibit a useful epitope ina fusion protein. The fusion of the large subunit of anthranilatesynthase (i.e., TrpE) murine Cyr61, and the fusion of gluttathioneS-transferase (i.e., GST) to murine Cyr61, have been used tosuccessfully raise antibodies against murine Cyr61. Yang et al. Inaddition, a wide variety of polypeptides, well known to those of skillin the art, may be used in the formation of Cyr61 fusion polypeptidesaccording to the invention.

[0122] More particularly, Yang reported a TrpE-Cyr61 fusion polypeptidethat was expressed from a recombinant clone constructed by cloning afragment of the murine cyr61 cDNA containing nucleotide 456 throughnucleotide 951 (encoding Cyr61 amino acids 93-379) into the SacI site ofthe pATH1 vector. Dieckman et al., J. Biol. Chem. 260:1513-1520 (1985).The recombinant construct was transformed into a bacterial host. e.g.,E. coli K12, and expression of the fusion protein was induced byaddition of 25 μg/ml indoleacrylic acid to growing cultures.Subsequently. cells were lysed and total cell lysate was fractionated byelectrophoresis on a 7.5% polyacrylamide gel. The fusion protein ofpredicted size was the only band induced by indoleacrylic acid; thatband was eluted from the gel and used as an antigen to immunize NewZealand White rabbits (Langshaw Farms) using techniques that arestandard in the art. Harlow et al. In addition to polyclonal antibodies.the invention comprehends monoclonal antibodies directed to such fusionproteins.

[0123] In other embodiments of the invention, recombinant antibodyproducts are used. For example. chimeric antibody products, “humanized”antibody products. and CDR-grafted antibody products are within thescope of the invention. Kashmiri et al., Hybridoma 14:461-473 (1995),incorporated herein by reference. Also contemplated by the invention areantibody fragments. The antibody products include the aforementionedtypes of antibody products used as isolated antibodies or as antibodiesattached to labels. Labels can be signal-generating enzymes, antigens,other antibodies, lectins, carbohydrates, biotin, avidin. radioisotopes,toxins, heavy metals, and other compositions known in the art,attachment techniques are also well known in the art.

[0124] Anti-Cyr61 antibodies are useful in diagnosing the risk ofthrombosis, as explained more fully in Example 20 below. In addition,anti-Cyr61 antibodies are used in therapies designed to prevent orrelieve undesirable clotting attributable to abnormal levels of Cyr61.Further, antibodies according to the invention can be attached to toxinssuch as ricin using techniques well known in the art. These antibodyproducts according to the invention are useful in deliveringspecifically-targeted cytotoxins to cells expressing Cyr61, e.g., cellsparticipating in the neovascularization of solid tumors. Theseantibodies are delivered by a variety of administrative routes. inpharmaceutical compositions comprising carriers or diluents. as would beunderstood by one of skill in the art.

[0125] Antibodies specifically recognizing Fisp12 have also beenelicited using a fusion protein. The antigen used to raise anti-Fisp12antibodies linked glutathione-S-transferase (GST) to the central portionof Fisp12 (GST-Fisp12), where there is no sequence similarity to Cyr61(O'Brien and Lau, 1992). A construct containing cDNA encoding aminoacids 165 to 200 of Fisp12 was fused to the glutathione-S-transferase(GST) coding sequence. This was done by using polymerase chain reaction(PCR) to direct synthesis of a fragment of DNA encompassing thatfragment of fisp12 flanked by a 5′ BamHI restriction site and a 3′ EcoRIrestriction site. The 5′ primer has the sequence5′-GGGGATCTGTGACGAGCCCAAGGAC-3′ (SEQ ID NO: 9) and the 3′ primer has thesequence 5′-GGGAATTCGACCAGGCAGTTGGCTCG-3′ (SEQ ID NO: 10). ForCyr61-specific antiserum, a construct fusing the central portion ofCyr61 (amino acids 163 to 229), which contains no sequence similarity toFisp12, to GST was made in the same manner using the 5′ primer5′-GGGGATCCTGTGATGAAGACAGCATT-3′ (SEQ ID NO: 11 ) and the 3′primer5′-GGGAATTCAACGATGCATTTCTGGCC-3′ (SEQ ID NO: 12). These weredirectionally cloned into pGEX2 T vector (Pharmacia-LKB, Inc.) and theclones confirmed by sequence analysis. The GST-fusion protein wasisolated on glutathione sepharose 4 B (Pharmacia-LKB, Inc.) according tomanufacturer's instructions, and used to immunize New Zealand whiterabbits. For affinity purifications, antisera were first passed througha GST-protein affinity column to remove antibodies raised against GST,then through a GST-Fisp12 or GST-Cyr61 protein affinity column toisolate anti-Fisp12 or anti-Cyr61 antibodies (Harlow et al., 1988).

[0126] These antibodies immunoprecipitated the correct size Fisp12protein product synthesized in vitro directed by fisp12 mRNA. Theantibodies are specific for the Fisp12 polypeptide and show nocross-reactivity with Cyr61.

[0127] Polyclonal antibodies recognizing CTGF are also known. U.S. Pat.No. 5.408,040, column 7, line 41, to column 9, line 63, incorporated byreference hereinabove, reveals an immunological cross-reactivity betweenPDGF and CTGF as described above.

EXAMPLE 12 Inhibitory peptides

[0128] Another embodiment of the present invention involves the use ofinhibitory peptides in therapeutic strategies designed to inhibit theactivity of the Cyr61 protein. One approach is to synthesize aninhibitory peptide based on the protein sequence of Cyr61. For example,a peptide comprising an amino acid sequence that is conserved betweenmurine Cyr61 (SEQ ID NO: 2) and human Cyr61 (SEQ ID NO: 4) competes withnative Cyr61 for its binding sites. This competition thereby inhibitsthe action of native Cyr61. For example, administration of an inhibitorypeptide by well-known routes inhibits the capacity of Cyr61 to influencethe cascade of events resulting in blood clots, the vascularization oftumors, or the abnormal vascularization of the eye (e.g., eye disorderscharacterized by vascularization of the retina or the vitreous humor),etc. In particular. an inhibitory peptide prevents Cyr61 from inhibitingthe action of Tissue Factor Pathway Inhibitor, or TFPI, as describedbelow.

[0129] In an embodiment of the invention, inhibitory peptides weredesigned to compete with Cyr61. These inhibitory peptides, like theantibodies of the preceding Example, exemplify modulators of Cyr61activity, as described in the context of a variety of assays for Cyr61activity that are disclosed herein. The peptide design was guided bysequence comparisons among murine Cyr61, Fisp12, and Nov (an avianproto-oncogene). The amino acid sequences of several members of thisfamily are compared in FIG. 1. These types of sequence comparisonsprovide a basis for a rational design for a variety of inhibitorypeptides. Some of these designed peptides, for example peptides spanningamino acids 48-68 (SEQ ID NO: 13). 115-135 (SEQ ID NO: 14), 227-250 (SEQID NO: 15), 245-270 (SEQ ID NO: 16). and 310-330 (SEQ ID NO: 17) of SEQID NO: 2. have been synthesized. A comparison of the murine Cyr61 aminoacid sequence and the human Cyr61 amino acid sequence reveals thatsimilar domains from the human protein may be used in the design ofpeptides inhibiting human Cyr61. In addition, sequence comparisons mayinvolve the human Cyr61 amino acid sequence; comparisons may alsoinclude the human homolog of Fisp12, Connective Tissue Growth Factor,also identified as a member of this protein family. O'Brien et al.(1992).

[0130] Inhibitory peptides may also be designed to compete with otherECM signalling molecules, e.g., Fisp12 or CTGF, for binding to theirrespective receptors. The design of inhibiting peptides is facilitatedby the similarity in amino acid sequences among the ECM signallingmolecules. In addition. inhibitory peptide design may be guided by oneor more of the methods known in the art for identifying amino acidsequences likely to comprise functional domains (e.g., hydrophilic aminoacid sequences as external/surface protein domains; sequences compatiblewith α-helical formation as membrane-spanning domains). These methodshave been implemented in the form of commercially available software,known to those of ordinary skill in the art. See e.g., theIntelligenetics Suite of Analytical Programs for Biomolecules.Intelligenetics, Inc., Mountain View, Calif. Using these approaches,inhibitory peptides interfering with the biological activity of an ECMsignalling molecule such as Cyr61, Fisp 12 or CTGF, may be designed.With the design of the amino acid sequence of an inhibitory peptide,production of that peptide may be realized by a variety of well-knowntechniques including, but not limited to, recombinant production andchemical synthesis. Exemplary peptides that have been shown tospecifically inhibit at least one biological activity of Cyr61 includepeptides exhibiting the “RGD” motif, or motif variants such as “RGDS,”“RGDSPK,” “GDR,” or “SGDR.” (Ruoslahti, et al., Science 238:491-497[1987], Ruoslahti, E., Ann. Rev. of Cell and Dev. Biol. 12:698-715[1996]) as described in Example 10 above.

EXAMPLE 13 Cell Adhesion

[0131] Another embodiment of the invention is directed to the use ofCyr61 to mediate cellular attachment to the extracellular matrix.Induction of cellular adhesion was investigated using murine Cyr61,fibronectin, and bovine serum albumin (BSA). Immunological 96-wellplates (Falcon brand) were coated with 50 μl of 0.1% BSA in PBS at 4° C.in the presence of 0-30 μg/ml concentrations of murine Cyr61 orfibronectin. After two hours exposure to the coating solution,non-diluted immune or pre-immune antisera (30 μl/well), oraffinity-purified anti-Cyr61 antibodies were added. For some wells, thecoating mixture was adjusted to 10 mM DTT or 100 mM HCl. After 16 hoursincubation, the coating solution was removed and the well surface wasblocked with 1% BSA in phosphate-buffered saline (PBS) for 1 hour atroom temperature. HUVE cells were plated in Ham's complete F12K medium[GIBCO-BRL, Inc.; Hams, Proc. Natl. Acad. Sci. (USA) 53:786 (1965)] at5×10³-10⁴ cells/well. Cycloheximide was added to 100 μg/ml immediatelybefore plating and monensin was added to 1 μM 14 hours before plating.After a 2-hour incubation at 37° C., the wells were washed with PBS andattached cells were fixed and stained with methylene blue. Theattachment efficiency was determined by quantitative dye extraction andmeasurement of the extract absorbance at 650 nm. Oliver et al., J. Cell.Sci. 92:513-518 (1989).

[0132] HUVE cells attached poorly to dishes treated with BSA alone. butadhered well to dishes coated with fibronectin. Murine Cyr61-coatedsurfaces also supported HUVE cell attachment in a dose-dependent manner.similar to fibronectin. For example, at 1 μg/ml. Cyr61 and fibronectinyielded A₆₅₀ values of 0.1. An A₆₅₀ value of 0.5 corresponded to theattachment of 6×10³ cells. At the other end of the tested concentrationrange. 30 μg/ml, Cyr61 yielded an A₆₅₀ of 0.8; fibronectin yielded anA₆₅₀ of 0.9. Cyr61 also promoted the attachment of NIH 3T3 cells. thoughless effectively than fibronectin. Cyr61-mediated cell attachment can beobserved as early as 30 minutes after plating, as visualized by lightmicroscopy.

[0133] The adhesion of HUVE cells on murine Cyr61-coated surfaces wasspecifically inhibited by anti-Cyr61 antiserum and by affinity-purifiedanti-Cyr61 antibodies, but not by pre-immune serum. In contrast,attachment of cells to fibronectin-coated dishes was not affected byeither the anti-Cyr61 antiserum or affinity-purified anti-Cyr61antibodies. These results show that enhancement of cell adhesion is aspecific activity of the Cyr61 protein. Furthermore. the Cyr61-mediatedcell attachment was insensitive to cycloheximide or monensin treatment,indicating that Cyr61 does not act by inducing de novo synthesis of ECMcomponents, stimulation of fibronectin, or collagen secretion. Rather,the data support the direct action of Cyr61 on cells in effectingadhesion. The Cyr61-mediated attachment of HUVE cells was completelyabolished by the presence of EGTA; however, attachment was restored bythe addition of CaCl₂ or MgSO₄ to the medium. These results indicatethat the interaction between Cyr61 and its cell surface receptorrequires divalent cations. consistent with the observations leading tothe identification of the α_(v)β₃ integrin as the Cyr61 receptordescribed in Example 10. above.

[0134] The ability of Cyr61 to promote cell adhesion, and the ability ofmolecules such as anti-Cyr61 antibodies to inhibit that process isexploited in an assay for modulators of cell adhesion. The assayinvolves a comparison of cell adhesion to surfaces, e.g., plastic tissueculture wells, that are coated with Cyr61 and a suspected modulator ofcell adhesion. As a control. a similar surface is coated with Cyr61alone. Following contact with suitable cells. the cells adhering to thesurfaces are measured. A relative increase in cell adhesion in thepresence of the suspected modulator, relative to the level of celladherence to a Cyr61-coated surface, identifies a promoter of celladhesion. A relative decrease in cell adhesion in the presence of thesuspected modulator identifies an inhibitor of cell adhesion.

[0135] The identification of a Cyr61 receptor led to the development ofa rapid and specific ligand-receptor assay (i.e., integrin bindingassay) for Cyr61. Monoclonal antibody LM609 (anti-α_(v)β₃ ) has beendescribed. Cheresh, 1987. Monoclonal antibody JBS5 (anti-fibronectinantibody) was purchased from Chemicon. Anti-human and anti-bovinevitronectin antisera were from Gibco BRL. HRP-conjugated goatanti-rabbit antibody was from KPL. RGDSPK peptide was from Gibco BRL;RGDS and SDGR peptides were from American Peptide Company. The peptidesfor functional assays were dissolved in PBS at 10 mg/ml and the pH wasadjusted to 7.5-8.0 with NaOH. Human plasma vitronectin was fromCollaborative Biomedical Products.

[0136] α_(v)β₃ integrin purification from HUVE cell lysates was done asdescribed in Pytela et al., Meth. Enzimol., 144:475-489 (1987). Briefly,10⁸ cells were lysed in 1 ml of PBS containing 1 mM CaCl₂, 1 mM MgCl₂,0.5 mM PMSF and 100 mM octylglucoside. The lysate was passed four timesthrough a 0.5 ml column containing RGDSPK Sepharose (prepared from thecyanogen bromide activated Sepharose CL 4 B as described in Lam, S. C.T., J. Biol. Chem., 267.5649-5655 (1992). The column was washed with 10ml of the lysis buffer and the bound protein was eluted with 2 ml of thesame buffer containing 1 mM RGDS peptide at room temperature. Theα_(v)β₃ integrin was dialyzed against PBS containing 1 mM CaCl₂, 1 mMMgCl₂, 5 mM octyl-glucoside and 0.1 mM PMSF with three changes of thedialysis buffer to remove the RGDS peptide. The protein was stored inaliquots at −70° C. The purity of the integrin was determined bySDS-PAGE under non-reducing conditions, followed by silver staining.Western blotting with anti-CD47 antibody showed that this α_(v)β₃integrin preparation does not contain any integrin-associated proteins.

[0137] The integrin binding assay was developed in accordance with thedisclosures in Brooks et al., Cell 85:683-693 (1996), and Lam, S. C.-T.(1992). Approximately 50 ng of the integrin in a total volume of 50 μlwere added per well of 96-well immunological Pro-Bind plates (Falcon)and incubated overnight at 4° C. Non-specific sites were blocked with 20mg/ml BSA in the same buffer and washed four times in that buffer.Treated plates were incubated with 1 μg/ml Cyr61 or 0.1 μg/mlvitronectin for 3 hours at room temperature. EDTA (5 mM), RGDS peptide(0.5 mM) and blocking antibodies were either preincubated with theimmobilized integrin for 1 hour before the addition of the proteinligand or added along with the ligand. The final dilution of the LM609ascites fluid was 1:200. Bound proteins were detected by specificpolyclonal antisera (anti-Cyr61 antiserum was diluted 1:500 andanti-vitronectin antiserum was diluted 1:1000 in PBS containing 1 mMCaCl₂ , 1 mM MgCl₂ , and 5 mg/ml BSA) followed by a secondaryantibody-horseradish peroxidase conjugate (1:20000 in the same buffer).Plates were rinsed four times with PBS containing 1 mM CaCl₂ and 1 mMMgCl₂ after each incubation. Horseradish peroxidase (HRP) was detectedwith an HRP immunoassay kit (Bio-Rad Laboratories). The calorimetricreaction was developed for 15-30 minutes at room temperature, stopped bythe addition of H₂SO₄, and the absorbance at 450 nm was measured. Thoseof ordinary skill in the art will understand that a variety of detectiontechniques could be employed in place of the enzyme-linked immunologicalapproach exemplified. For example, other labels such as radiolabels,fluorescent compounds and the like could be bound, e.g., covalently, toan antibody or other agent recognizing the peptide of interest such asCyr61.

[0138] The results of integrin binding assays showed that vitronectinand Cyr61 bound to the immobilized integrin. Further. both Cyr61 andvitronectin binding to α_(v)β₃ were saturable. The concentration ofCyr61 at which saturation was reached was significantly higher than theconcentration of vitronectin required for saturation. This differencemay reflect a lower affinity of α_(v)β₃ for Cyr61 compared tovitronectin, which is in agreement with the results of cell adhesionassays, which show that HUVE cells adhere to vitronectin and, moreweakly, to Cyr61. in a concentration-dependent manner (see below). Thespecificity of the interaction was addressed by blocking the ligandbinding site of the integrin using any one of several techniques,including divalent cation deprivation, RGDS peptide competition. andLM609 antibody inhibition. The interaction of both proteins (Cyr61 andvitronectin) with α_(v)β₃ was inhibited by EDTA, the RGDS peptide, andthe LM609 antibody. These properties of the Cyr61 interaction withα_(v)β₃ were also in agreement with the results of the cell adhesionassay and indicated that HUVE cell adhesion to Cyr61 was mediated by thedirect interaction of Cyr61 with the α_(v)β₃ integrin.

[0139] In addition, Cyr61 induces focal adhesion, i.e., cell surfacefoci for cytoskeletal attachments. Focal adhesion is effected by cellsurface protein complexes or clusters. These protein clusters arecomplex, including a variety of receptors from the integrin family, anda variety of protein kinases. The induction of focal adhesion by Cyr61is reflected in the capacity of Cyr61 to induce particular members ofthese cell surface protein clusters. For example, Cyr61 induces thephosphorylation of Focal Adhesion Kinase, a 125 kDa polypeptide, andPaxillin, another protein known to be involved in the focal adhesioncell surface protein complexes. Moreover, indirect immunofluorescencestudies have shown that Cyr61 is bound to a receptor (see above) infocal adhesive plaques. The plaques, in turn, are characteristic offocal adhesion protein complexes. Focal Adhesion Kinase, Paxillin, andα_(v)β₃ Integrin are co-localized to the focal adhesion plaques producedby focal adhesion complex formation induced by Cyr61. These focaladhesion protein complexes bind Cyr61 at the cell surface; the complexesalso attach internally to the cytoskeleton. Therefore, murine Cyr61, andhuman Cyr61 (see below). are, in part, adhesion molecules, acharacteristic distinguishing Cyr61 from conventional growth factors.Those of skill in the art will also recognize that the α_(v)β₃ integrincan be used, in conjunction with Cyr61. to screen for modulators ofCyr61 binding to its receptor. In one embodiment, the integrin isimmobilized and exposed to either (a) Cyr61 and a suspected modulator ofreceptor binding; or (b) Cyr61 alone. Subsequently, bound Cyr61 isdetected. e.g., by anti-Cyr61 antibody that is labeled using techniquesknown in the art, such as radiolabelling, fluorescent labelling. or theuse of enzymes catalyzing colorimetric reactions. A promoter of Cyr61binding to its receptor would increase binding of Cyr61 (and aninhibitor would decrease Cyr61), relative to the binding by Cyr61 alone.

[0140] In another embodiment of the invention, the effect of murineCyr61 on cell morphogenesis was assessed by a cell spreading assay.Polystyrene Petri dishes were coated with 2 ml of a 10 μg/ml solution ofCyr61 or fibronectin in PBS with 0.1% BSA and treated as describedabove. A third plate was treated with BSA and served as a control. Eachdish received 7×10⁶ cells and was incubated for 2 hours. Cell spreadingwas analyzed by microscopy at 100-fold magnification. The resultsindicate that murine Cyr61 induces HUVE cell spreading to approximatelythe same extent as fibronectin. The efficient attachment (see above) andspreading of cells on murine Cyr61-coated substrates indicated thatCyr61 may interact with a signal-transducing cell surface receptor,leading to a cascade of cytoskeletal rearrangements and possibleformation of focal contacts. Consequently, Cyr61 and Cyr61-relatedpolypeptides may prove useful in controlling cell adhesion, e.g., thecell adhesion events that accompany metastasizing cancer cells, organrepair and regeneration, or chondrocyte colonization of prostheticimplants, discussed below.

[0141] In contrast to mouse Cyr61 which mediated both HUVE cellattachment and migration, hCyr61 was found to mediate cell adhesion butnot spreading of HUVE cells. Immunological plates (96-well ProBind assayplates, Falcon) were coated with 0.1-30 μg/ml hCyr61. fibronectin (GibcoBRL) or vitronectin (Gibco BRL) in phosphate-buffered saline (PBS)containing 0.1% protease-free BSA (Sigma) for 16 hrs at 4° C. The wellswere blocked with 1% BSA in PBS for 1 hr at room temperature and washedwith PBS. HUVE cells were harvested with 0.02% EDTA in PBS. washed twicewith serum-free F12 medium and resuspended in serum-free F12. In someexperiments, fbs was added to 5-10%. Also, in experiments involvingvitronectin-coated plates, endogenous vitronectin was removed from fbsby immunoaffinity chromatography using bovine polyclonalanti-vitronectin antibodies (Gibco). Norris et al., J. Cell Sci.95:255-262 (1990). Cells were plated at 10⁴ cells/well. After 2 hours,cells were fixed with 4% paraformaldehyde, stained with methylene blueand quantified as described. Oliver et al., J. Cell Sci. 92:513-518(1989).

[0142] Under serum-free conditions. hCyr61 mediated cell attachment butnot spreading of HUVE cells. Attachment of HUVE cells to hCyr61-coatedplates was enhanced by inclusion of serum in the culture medium. In thepresence of serum. HUVE cells attached and spread on hCyr61 in a mannersimilar to that seen on fibronectin. Human Cyr61 supported HUVE celladhesion in a dose-dependent manner both under high-serum (10%) andlow-serum (0.5%) conditions. However. in the presence of 10% fbs, themaximal proportion of the cells attaching at a lower concentration ofhCyr61, and the proportion of the cells attached. was higher. HumanCyr61 was also found to cooperate with vitronectin in promoting HUVEcell adhesion and spreading. Two major cell-adhesive proteins found inmammalian sera are fibronectin and vitronectin, also known as “serumspreading factor.” For review, see Felding-Habermann et al., Curr. Opin.Cell Biol. 5:864-868 (1993). Cell attachment. spreading and growth ontissue-culture plastic depended upon vitronectin. rather thanfibronectin. in serum for the following reasons: (1) considerabledepletion of fibronectin in the batches of fbs due to “clotting” at 4°C.; and (2) inability of fibronectin to efficiently coat the plastic inthe presence of an excess amount of other serum proteins. In contrast.vitronectin coated the plastic surfaces efficiently under the sameconditions.

[0143] The ability of HUVE cells to adhere to hCyr61-coated plates inthe presence of mock-immunodepleted fbs and serum immunodepleted withanti-bovine vitronectin antibodies were compared. HUVE cells adhered tohCyr61-coated surfaces significantly better in the presence of solublevitronectin or mock-immunodepleted fbs than they did in the presence ofserum-free medium or medium supplemented with vitronectin-immunodepletedfbs. The addition of vitronectin (30 μg/ml) tovitronectin-immunodepleted serum restored the ability of HUVE cells toadhere and spread on hCyr61-coated plates to the same level observedwhen whole serum was used in the cell attachment assay. Furthermore,soluble vitronectin alone. at a concentration equal to its level in 10%serum (30 μg/ml). restored the level of cell adhesion and spreading tothe level found in the presence of 10% serum. Thus, vitronectin is anecessary and sufficient serum component contributing to HUVE celladhesion and spreading on hCyr61-coated plastic surfaces. Controlstudies showed that the effect of vitronectin was not due to itspreferential retention on the plastic dish surfaces in the presence ofhCYR61.

[0144] Additionally, HUVE cell attachment and spreading in the presenceof an increasing quantity of vitronectin was examined. The solutions forcoating the dishes contained increasing amounts of vitronectin (0-10μg/ml) with a fixed amount of hCyr61 (10 μ/ml). The results indicatedthat more cells adhered to plates coated with the two proteins thanwould have been expected by adding the individual adhesive capacities ofvitronectin and hCyr61. This non-additive increase of adhesion in thepresence of vitronectin and hCyr61 was not due to higher amounts ofvitronectin absorbed on the plastic. ELISA assay with anti-humanvitronectin antibodies showed that the amount of vitronectin adsorbed toplastic dishes exposed to the vitronectin/hCyr61 mixture did not exceedthat of vitronectin alone by more than 20%. This difference isinsufficient to explain the observed difference in cell adhesion (3-5fold in different experiments). In addition. a higher proportion of HUVEcells also adhered to the mixture of proteins when the coating solutioncontained diluted vitronectin (2.5 μg/ml) than were found to adhere todishes coated with higher concentrations of pure vitronectin (10 μg/ml)or pure liCyr61 (10 μg/ml). Thus, vitronectin and hCyr61 functionallycooperate and exert a synergistic effect on HUVE cell adhesion.

[0145] The capacity of Fisp12 to affect cell adhesion was alsoinvestigated. Fisp12 cell attachment assays were performed essentiallyas described (Oliver et al. 1989). 96-well immunological plates werecoated for 16 hours at 4° C. with 20 μg/ml Cyr61, Fisp12 or fibronectin(Gibco BRL) in PBS containing 0.1 mg/ml BSA and blocked with 10 mg/mlBSA for 1 hour at room temperature. HUVE cells were plated at 10⁴cells/well in F12K media with 10% FBS (Hyclone Laboratories, Inc.,Logan, Utah): NIH 3T3 fibroblasts were plated at 3×10⁴ cells/well andMv1Lu cells were plated at 5×10⁴ cells/well in minimal essential medium(MEM) with 10% FBS. After 1 hour incubation cells were fixed, stainedwith methylene blue and quantified as described (Oliver et al. 1989).Cell spreading was examined on cells plated on 100 mm polystyrene petridishes coated with 2.5 ml of a 20 μg/ml solution of Cyr61, Fisp12 orfibronectin. 10⁷ cells were plated on each dish and cell spreading wasanalyzed 90 min. after plating by microscopy at 100×magnification.

[0146] The results indicated that Fisp12, as well as Cyr61, when coatedon plastic dishes, promoted the attachment of three different celltypes: HUVE cells, NIH 3T3 fibroblasts, and mink lung epithelial (Mv1Lu)cells. These cells attached poorly to uncoated plastic dishes or plasticdishes coated with bovine serum albumin, but attached significantlybetter to dishes coated with either fibronectin, Cyr61, or Fisp12. Theability of either Cyr61 or Fisp12 to mediate cell attachment iscomparable to that of fibronectin for all three cell types. While theability of Cyr61 to mediate cell attachment was previously demonstratedfor fibroblasts and endothelial cells (Kireeva et al. 1996), thesestudies show cell attachment activity for both Fisp12 and Cyr61 inepithelial cells in addition to endothelial cells and fibroblasts.

[0147] Like cell attachment to fibronectin and Cyr61 (Kireeva et al.,1996). Fisp 12-mediated cell attachment was inhibited when EDTA wasadded to the culture medium. This inhibition was completely abolished bythe addition of excess MgCl₂, indicating a requirement for divalentcations in Fisp12-mediated cell attachment. In addition to cellattachment, Fisp12 also promotes cell spreading. Similar cell spreadingwas found when NIH 3T3 cells were plated on dishes coated with eitherfibronectin, Cyr61, or Fisp12, but not BSA. Endothelial and epithelialcells also spread when plated on fibronectin, Cyr61. or Fisp12.

EXAMPLE 14 Migration of Fibroblasts

[0148] Cyr61 also affects chondrocytes, e.g., fibroblasts involved inskeletal development. In particular, Cyr61 influences the development,and perhaps maintenance. of cartilage, in contrast to the variety ofgrowth-related proteins that exclusively influence development andmaintenance of the bony skeleton. The chemotactic response of NIH 3T3cells to murine Cyr61 was examined using a modified Boyden chamber(Neuroprobe Inc., catalog no. AP48). Grotendorst, Meth. Enzymol.147:144-152 (1987). Purified Cyr61 protein was serially diluted in DMEMcontaining bovine serum albumin (BSA; 0.2 mg/ml) and added to the lowerwell of the chamber. The lower well was then covered with acollagen-coated polycarbonate filter (8 μm pore diameter; NucleoporeCorp., Pleasanton, Calif.). Cells (6×10⁴) were then loaded into theupper well. After 5 hours incubation (10% CO₂, 37° C.), the filter wasremoved and the cells were fixed and stained using Wright-Giemsa stain(Harleco formulation; EM Diagnostic Systems, Gibbstown, N.J.). Cellsfrom the upper surface of the filter were then removed by wiping with atissue swab. The chemotactic response was determined by counting thetotal number of migrating cells detected in ten randomly selectedhigh-power microscopic fields (400-fold magnification) on the lowersurface of the filter. Duplicate trials were performed for eachexperiment and the experiment was repeated three times to ensurereproducibility of the data.

[0149] NIH 3T3 cells responded to Cyr61 as a chemotactic factor in adose-dependent manner in the Boyden chamber assay. Without Cyr61,approximately 4.8 cells had migrated per high-power field. In thepresence of 0.5 μg/ml murine Cyr61, about 5.2 cells were found in eachfield. As the concentration of Cyr61 was raised to 1, 5 and 10 μg/ml,the average number of migrating cells detected per field rose to 7.5,18.5, and 18.7. Thus murine Cyr61 acts as a chemoattractant forfibroblasts. The optimal concentration for the chemotactic activity ofCyr61 is 1-5 μg/ml in this assay; this concentration range is consistentwith the reported ranges at which other ECM molecules provide effectivechemotactic stimulation. For example, Thrombospondin. at 5-50 μg/ml. hasa chemotactic effect on endothelial cells (Taraboletti et al., J. CellBiol. 111: 765-772 (1990): fibronectin also functions as a chemotacticagent at 1-30 μg/ml (Carsons et al., Role of Fibronectin in RheumaticDiseases, in Fibronectin [Mosher. ed., Academic Press 1989]; Carsons etal., Arthritis. Rheum. 28:601-612 [1985]) as determined using similarBoyden chamber assays. The human Cyr61 polypeptide may be used tochemoattract fibroblasts in a manner analogous to murine Cyr61. HumanCTGF has also been reported to induce the migration of non-humanmammalian cells such as NIH 3T3 cells (mouse fibroblasts) and BASM cells(bovine aortic smooth muscle cells), as described in U.S. Pat. No.5,408,040, column 7, line 65 to column 11, line 7, incorporated hereinby reference.

[0150] In an alternative embodiment, an assay for modulators of cellmigration, such as the migration of chondrocytes, involves a combinationof a suspected modulator of cell migration and Cyr61 being added to thelower well of a Boyden chamber. As a control, Cyr61 is separately addedto the lower well of another Boyden chamber. Relative cell migrationsare then measured. An increase in cell migration in the presence of thesuspected modulator relative to cell migration in response to Cyr61alone identifies a promoter of chondrocyte cell migration, while arelative decrease in cell migration in the presence of the suspectedmodulator identifies an inhibitor.

EXAMPLE 15 Migration of Endothelial Cells—In Vitro Assays

[0151] The end product of in vitro angiogenesis is a well-definednetwork of capillary-like tubes. When cultured on gel matrices, e.g.,collagen, fibrin, or Matrigel gels, endotlielial cells must first invadethe matrix before forming mature vessels. (Matrigel is a complex mixtureof basement membrane proteins including laminin, collagen type IV,nidogen/entactin, and proteoglycan heparin sulfate, with additionalgrowth factors. Kleinman et al., Biochem. 25:312-318 (1986). Theinvasive structures are cords which eventually anastomose to form thevessel-like structures. The angiogenic effect of human Cyr61 onconfluent monolayers of human umbilical vein endothelial cells isassessed by seeding the cells onto three-dimensional collagen or fibringels, in the presence or absence of Cyr61. HUVE cells do notspontaneously invade such gels but do so when induced by agents such astumor promoters.

[0152] Collagen gels were prepared by first solubilizing type I collagen(Collaborative Research, Inc., Bedford, Mass.) in a sterile 1:1000 (v/v)dilution of glacial acetic acid (300 ml per gram of collagen). Theresulting solution was filtered through sterile triple gauze andcentrifuged at 16,000×g for 1 hour at 4° C. The supernatant was dialyzedagainst 0.1×Eagle's Minimal Essential Medium (MEM; GIBCO-BRL, Inc.) andstored at 4° C. Gels of reconstituted collagen fibers were prepared byrapidly raising the pH and ionic strength of the collagen solution. ThepH and ionic strength adjustments were accomplished by quickly mixing 7volumes of cold collagen solution with one volume of 10×MEM and 2volumes of sodium bicarbonate (11.76 mg/ml) in a sterile flask. Thesolution was kept on ice to prevent immediate gelation. The cold mixturewas dispensed into 18 mm tissue culture wells and allowed to gel for 10minutes at 37° C.

[0153] Fibrin gels were prepared by dissolving fibrinogen (SigmaChemical Co. St. Louis, Mo.) immediately before use in calcium-free MEMto obtain a final concentration of 2.5 mg of protein/ml. Clotting wasinitiated by rapidly mixing 1.35 ml of fibrinogen solution with 15 μl of10×MEM containing 25 U/ml thrombin (Sigma Chemical Co.) in a plastictube. The mixture was transferred immediately into 18 mm tissue culturewells and allowed to gel for about 2 minutes at 37° C.

[0154] In some wells. Cyr61 was mixed into the gel matrix beforegelation (final concentration 10 μg/ml), while in other wells, Cyr61 wasnot in the gel matrix but was added as part of the nutrient medium(similar range of concentrations as in the matrix) after the cellsreached confluency. HUVE cells were seeded onto the gel matrix surfaceat 5×10⁴ cells per well in Ham's F12K medium [GIBCO-BRL. Inc.]containing 10% fetal bovine serum, 100 μg/ml heparin, and 30 μg/mlendothelial cell growth factor. When the cells reached confluency, themedium was removed, the cells were rinsed with PBS, and fresh mediumwithout endothelial cell growth factor was supplied. Some culturesreceived purified recombinant Cyr61, while others received Cyr61 andpolyclonal anti-Cyr61 antibodies. Thus, the variety of cultures atconfluency included: a) cultures with no Cyr61; b) cultures with Cyr61within the matrix: c) cultures with Cyr61 supplementing the medium; andd) cultures with Cyr61 supplementing the medium along with polyclonalanti-Cyr61 antibodies.

[0155] Invasion of the gel matrix was quantified about 4-7 days aftertreatment of the confluent cultures. Randomly selected fields measuring1.0 mm×1.4 mm were photographed in each well under phase-contrastmicroscopy with a Zeiss Axiovert inverted photomicroscope. Photographswere taken at a single level beneath the surface monolayer. Invasion wasquantified by measuring the total length of all cell cords thatpenetrated beneath the surface monolayer. Results were expressed as themean length in microns per field for at least 3 randomly selected fieldsfrom each of at least 3 separate experiments.

[0156] In order to examine the network of cords within the matrix forcapillary-like tube formation, cultures were fixed in situ overnightwith 2.5% glutaraldehyde and 1% tannic acid in 100 mM sodium cacodylatebuffer, pH 7.4. Cultures were then washed extensively in 100 mM sodiumcacodylate buffer, pH 7.4. The gels were cut into 2 mm×2 mm fragments,post-fixed in 1% osmium tetroxide in veronal acetate buffer (to minimizetissue swelling; see Havat, in Principles and Techniques of ElectronMicroscopy: Biological Applications 1:38 [Litton Educational Publishing,Inc. 1970]) for 45 minutes, stained en bloc with 0.5% uranyl acetate inveronal buffer for 45 minutes, dehydrated by exposure to a gradedethanol series, and embedded in Epon in flat molds. Semi-thin sectionswere cut perpendicular to the culture plane with an ultramicrotome,stained with 1% toluidine blue, and photographed under transmitted lightusing an Axiophot photomicroscope (Zeiss).

[0157] In an alternative embodiment, a suspected modulator ofangiogenesis is combined with Cyr61 and the combination is added before,or after, formation of a gel. In this embodiment, a control isestablished by using Cyr61 alone. The migration of cells in response tothe suspected modulator and Cyr61 is then compared to the migration ofcells in response to Cyr61 alone. A promoter or positive effector willincrease cell migration while an inhibitor or negative effector willdecrease cell migration.

[0158] In an alternative in vitro assay for angiogenic activity. anassay for endothelial cell migration was developed. This chemotaxisassay has been shown to detect the effects of Cyr61 concentrations onthe order of nanograms per milliliter. Primary Human MicrovascularEndothelial Cells (HMVEC PO51: Clonetics. San Diego, Calif.) weremaintained in DME with 10% donor calf serum (Flow Laboratories, McLean,Va.) and 100 μg/ml endothelial cell mitogen (Biomedical TechnologiesInc., Stoughton, Mass.). The cells were used between passages 10 and 15.To measure migration. cells were starved for 24 hours in DME containing0.1% BSA, harvested, resuspended in DME with 0.1% BSA. and plated at1.75×10⁴ cells/well on the lower surface of a gelatinized 0.5 μm filter(Nucleopore Corporation, Pleasanton, Calif.) in an inverted modifiedBoyden chamber. After 1-2 hours at 37° C., during which time the cellswere allowed to adhere to the filter, the chamber was reverted to itsnormal position. To the top well of separate chambers, basic FibroblastGrowth Factor (a positive control), Cyr61, or a negative controlsolution (conditioned medium known to lack chemoattractants or DME plusBSA, see below) was added at concentrations ranging from 10 ng/nil to 10μg/ml. Chambers were then incubated for 3-4 hours at 37° C. to allowmigration. Chambers were disassembled. membranes fixed and stained, andthe number of cells that had migrated to the top of the filter in 3high-powered fields was determined. Tolsma et al., J. Cell. Biol.122:497-511 (1993) (incorporated by reference), and references citedtherein. DME with 0.1% BSA was used as a negative control and eitherbFGF (10 ng/ml) or conditioned media from angiogenic hamster cell lines(20 μg/ml total protein) were used as positive controls. Rastinejad etal., Cell 56:345-355 (1989). Each sample was tested in quadruplicate(test compound such as Cyr61, positive control, conditioned medium as anegative control. and DME plus BSA as a negative control) in a singleexperiment; experiments were repeated at least twice.

[0159] To allow comparison of experiments performed on different days.migration data is reported as the percent of maximum migration towardsthe positive control, calculated after subtraction of backgroundmigration observed in the presence of DME plus BSA. Test compounds thatdepressed the random movement of endothelial cells showed a negativevalue for the percent migration. Very high concentrations ofthrombospondin (TSP) caused endothelial cells to detach from themembrane. Detachment was detected by counting cells on the lower face ofthe membrane. When cell loss exceeded 10%. the number of migrated cellswas corrected for this loss. The results indicate that 0.01-10 μg/mlbFGF induced the migration of a constant 92 cells per three high-poweredmicroscope fields Migration in the presence of Cyr61 revealed a greaterdependence on concentration. At 10 ng/ml, Cyr61 induced an average of 64cells to migrate per three high-powered fields examined. At 100 ng/mlCyr61, approximately 72 cells were found in three fields; at 1 μg/mlCyr61, a peak of 87 cells had migrated; at approximately 7 μg/ml Cyr61,about 61 cells were observed; and at 10 μg/ml Cyr61, approximately 57cells were found to have migrated. The negative control revealed aconstant basal level of endothelial cell migration of 53 cells per threehigh-powered microscope fields. In addition to these results, there is aperfect correlation of the results from this in vitro assay and theresults from the in vivo cornea assay, described below.

[0160] To monitor toxicity. endothelial cells were treated with each ofthe tested compounds at a range of concentrations, under conditionsidentical to those used in the migration assay. Cells were then stainedwith Trypan blue and cells excluding Trypan blue were counted. Theresults showed that cells remained viable and that the inhibition ofmigration could not be attributed to toxicity. Where relevant,endothelial cells were pretreated for 36-48 hours with peptides at 20 μMin DME with 0.1% BSA before use in the migration assays. Toxicity wasalso tested over these time frames and found to be negligible.

[0161] The ability of Cyr61 to induce matrix invasion and tube formationby HUVE cells. as well as the ability of Cyr61 to induce humanmicrovascular endothelial cells to migrate. demonstrates the angiogenicproperties of this protein. It is anticipated that other members of theECM signalling molecule family of cysteine-rich proteins, such as Fisp12and CTGF. have similar properties that may be used in methods of theinvention for screening for, and modulating, angiogenic conditions. Inparticular. one of ordinary skill in the art understands that an invitro assay for angiogenic inhibitors involves the assay describedabove, including an effective amount of Cyr61, with and without thecandidate inhibitor.

EXAMPLE 16 Migration of Endothelial Cells—An In Vitro Assay ForAngiogenesis Inhibitors

[0162] The inclusion of an effective amount of an ECM signallingmolecule, such as Cyr61 in the in vitro migration (i.e., chemotaxis)assay described in the preceding Example, provides an assay designed todetect inhibitors of ECM signalling molecules and angiogenesis. Becauseof the crucial role of neovascularization in such processes as solidtumor growth and metastasis. the development of assays to detectcompounds that might antagonize these processes would be useful.

[0163] The above-described in vitro migration assay was adapted toinclude an ECM signalling molecule, Cyr61. Cyr61 was included at 1μg/ml, which was found to be the optimal dosage in titration studies. Asin the preceding Example, human microvascular endothelial cells(Clonetics) were used. In one series of assays, several carbohydratesand carbohydrate derivatives were analyzed. These compounds included 10mM mannose, 10 mM mannose-6-phosphate, and 10 mM galactose. Results ofthese assays showed that Cyr61 plus mannose yielded approximately 73cells per set of three high-powered microscope fields (see above). Cyr61plus galactose induced the migration of approximately 74 cells per setof three high-powered fields. However, Cyr61 plus mannose-6-phosphateyielded approximately 2 migrating cells for each set of threehigh-powered fields examined. Control experiments demonstrate that theinhibition of Cyr61 activity by mannose-6-phosphate is specific.

[0164] The angiogenic activity of basic FGF (10 ng/ml) was also tested.as described above, with and without mannose-6-phosphate. In thepresence of 10 mM mannose-6-phosphate, bFGF induced 51 cells per set ofthree high-powered fields to migrate; in its absence, bFGF induced themigration of approximately 52 cells. However, when either Cyr61 orInsulin Growth Factor II (IGF-II) were tested, mannose-6-phosphatereduced the number of migrating cells from approximately 48 or 47 cells.respectively, to approximately 12 or 11 cells. respectively. The effectof mannose-6-phosphate on IGF II activity was anticipated becausemannose-6-phosphate is known to compete with IGF II for their commonreceptor (the IGF II receptor). Thus, mannose-6-phosphate specificallyinhibits the chemotactic activity of Cyr61 on human endothelial cells.Moreover, because there is an essentially perfect correlation betweenthe in vitro migration assay and the in vivo angiogenesis assay,described below, mannose-6-phosphate has been identified as an inhibitorof angiogenesis based on the results of the assay disclosed herein.Accordingly, the invention contemplates a method of inhibitingangiogenesis comprising the step of administering an inhibitor theangiogenic activity of Cyr 61 such as mannose-6-phosphate. Assays suchas that described above may also be used to screen for other inhibitorsof angiogenesis which may be useful in the treatment of diseasesassociated with angiogenesis such as cancer. and diseases of the eyewhich are accompanied by neovascularization.

[0165] In an embodiment of the invention, a method of screening formodulators of angiogenesis involves a comparative assay. One set ofconditions involves exposure of cells to a combination of Cyr61 and asuspected modulator of cell migration. As a control, a parallel assay isperformed that exposes cells to Cyr61 alone. A promoter of cellmigration elevates the rate of in vitro cell migration relative to therate of migration in the presence of Cyr61 alone, the converse is truefor an inhibitor of the chemoattracting ability of Cyr61.

EXAMPLE 17 Migration of Endothelial Cells—An In Vivo Assay

[0166] An in vivo assay for endothelial cell migration has also beendeveloped. In general, the assay protocol is consistent with thedisclosure of Tolsma et al. 1993. To assess angiogenesis associated withthe formation of granulation tissue (i.e., the newly formed,proliferative, fibroblastic dermal tissue around wounds during healing),sponge implants were used as previously described (Fajardo, et al., Lab.Invest. 58:718-724 [1988]). Polyvinyl-alcohol foam discs (10-mmdiam×1-mm thick) were prepared by first removing a 2-mm diameter centralcore of sponge. PBS or an RGDS peptide (other possible test compoundsinclude fragments of Cyr61, RGDS peptide. small molecules such asmannose-6-phosphate) at 100 μM were added to the sponge core which wasthen coated with 5 μl of sterile Hydron (Interferon Sciences, NewBrunswick. N.J.). After solidifying, the coated core was returned to thecenter of the sponge which was then covered on both sides with 5 μmfilters and secured in place with glue (Millipore Corp., Bedford,Mass.). One control and one test disc were then implanted subcutaneouslyin the lower abdomen of anesthetized Balb/c female mice wheregranulation tissue could invade the free perimeter of the disc. Woundswere closed with autoclips and animals left undisturbed untilsacrificed.

[0167] Quantitative estimates of thymidine incorporation in situ intoendothelial cells in the discs were obtained as previously described(Polverini, et al, J. Immunol. 118:529-532 [1977]). Sponge implants wereevaluated at days 5, 7, 10, and 14 after implantation. Thirty minutesbefore sacrifice, mice were injected with a solution containing[³H]-thymidine in saline (specific activity 6.7 Ci/mM; New EnglandNuclear/Du Pont, Wilmington, Del.) to a level of 1 μCi per gram of bodyweight. Sponges were removed and facially embedded to provide a uniformsection of the entire circumference. Tissues were fixed in 10% neutralbuffered formalin, dehydrated through a graded series of alcohols, andembedded in glycol methacrylate (Polysciences. Miles.

[0168] Ill.). Autoradiograms were prepared by dipping sections mountedon acid-cleaned glass slides into NTB type 2 emulsion (Eastman Kodak).After exposure for 4 weeks at 4° C., autoradiographs were developed inhalf strength D-19 developer. fixed in Kodak Rapid Fixer, and stainedwith hematoxylin and eosin. Quantitation of endothelial cell labelingwas performed by counting all endothelial cells that lined capillariesand venules extending from the periphery to the center of the sponge byrectilinear scanning under oil immersion (×1.000). A total of 500-700endothelial cells were counted in each of two sponges containing eitherPBS, TSP, or peptide fragments (i.e., thrombospondin fragments). Cellswere considered labeled if five or more grains were detected over thenucleus. The percentage of labeled cells was calculated and a chi-squareanalysis of data derived from control and experimental sponges wasperformed.

[0169] The results of the foregoing assay established thatthrombospondin fragments could inhibit the process of angiogenesis. Moregenerally, one of ordinary skill in the art would appreciate that thescope of the present invention extends to such in vivo assays forsuspected modulators of ECM signalling molecule activities, such as thechemotactic ability of Cyr61 to induce cell migration. As with otherassays of the invention, a comparative assay involves exposure of cells,in vivo, to a sponge laden with Cyr61 in the presence or absence of asuspected modulator of Cyr61 activity. Following implantation,incubation. and removal, the relative rates of cell migration aredetermined. A promoter of Cyr61 activity will increase the rate of cellmigration relative to cell migration induced by Cyr61 alone; aninhibitor will decrease the rate of cell migration relative to the levelascribable to Cyr61 alone.

EXAMPLE 18 Mitogen Potentiation

[0170] In another aspect of the invention, murine Cyr61 enhanced themitogenic effect of growth factors on fibroblasts and endothelial cells.When NIH 3T3 fibroblasts or HUVE cells were treated with anon-saturating dose of either basic Fibroblast Growth Factor (bFGF) orPlatelet-Derived Growth Factor (PDGF-BB), the addition of murine Cyr61significantly increased the incorporation of radiolabeled thymidinecompared to cells treated with the growth factors alone. The thymidineincorporation assay is a standard technique for determining whethercells are actively growing by assessing the extent to which the cellshave entered the S phase and are synthesizing DNA. The Cyr61 enhancementof bFGF- or PDGF-BB-induced thymidine incorporation was dose dependent,requiring a minimum concentration of 0.5 -1.0 μg/ml of recombinantprotein for either cell type. The enhancement of DNA synthesis by Cyr61was inhibited by the addition of specific anti-Cyr61 antiserum.

[0171] More specifically, NIH 3T3 fibroblast cells were plated on24-well plates at 3×10⁴ cells/well and grown in DMEM with 10% fetalbovine serum (Intergen Co., Purchase, N.Y.) for 3-4 days and incubatedwith medium containing 0.2% FBS for the following 48 hours. Thefollowing compounds, at the parenthetically noted final concentrations,were then added to the plated cells in fresh DMEM containing 0.2% fbsand [³ H]-thymidine 1 μCi/ml final concentration: ICN Biomedicals, Inc.,Costa Mesa, Calif.): bFGF (15 ng/ml), PDGF-BB (30 ng/ml). and murineCyr61 (0.5-5 μg/ml). These compounds were added to individual platesaccording to the following pattern: 1) no supplementation: 2) murineCyr61; 3) bFGF; 4) murine Cyr61 and bFGF; 5) PDGF-BB: and 6) murineCyr61 and PDGF. After 18-20 hours of incubation, cells were washed withPBS and fixed with 10% trichloroacetic acid. DNA was dissolved in 0.1 NNaOH and thymidine incorporation was determined. The results indicatedthat murine Cyr61, in the absence of a growth factor, did not stimulateDNA synthesis as measured by tritiated thymidine incorporation. Withoutany supplements. 3T3 cells incorporated approximately 1.8×10⁴ cpm of[³H]-thymidine, in the presence or absence of Cyr61. Cells exposed tobFGF alone incorporated about 1.2×10⁵ cpm: cells contacting bFGF andmurine Cyr61 incorporated 2×10⁵ cpm. Cells receiving PDGF-BBincorporated about 1.2×10⁵ cpm; and cells exposed to PDGF-BB and murineCyr61 incorporated approximately 2.4×10⁵ cpm. Therefore. murine Cyr61did not function as a mitogen itself, but did potentiate the mitogenicactivity of bFGF and PDGF-BB, two known growth factors.

[0172] The ability of murine Cyr61 to potentiate the mitogenic effect ofdifferent levels of bFGF also revealed a threshold requirement for thegrowth factor. Human umbilical vein endothelial cells were platedessentially as described above for 3T3 cells and exposed to a constantamount of murine Cyr61: controls received no Cyr61. Different plateswere then exposed to different levels of bFGF. comprising a series ofbFGF concentrations ranging from 0-10 ng/ml. Following culture growth inthe presence of [³H]-thymidine for 72 hours, cells exposed to 0-0.1ng/ml of bFGF exhibited a baseline level of thymidine incorporation(approximately 4×10² cpm), in the presence or absence of Cyr61. At 1ng/ml bFGF, however, HUVE cells increased their thymidine incorporationin the presence of bFGF to 6×10² cpm; in the presence of 1 ng/ml bFGFand murine Cyr61, HUVE cells incorporated 1.3 ×10³ cpm. At 10 ng/mlbFGF. cells exposed to bFGF incorporated about 1.8×10³ cpm thymidine:cells receiving 10 ng/ml bFGF and Cyr61 incorporated approximately6.1×10³ cpm.

[0173] The capacity of murine Cyr61 to potentiate the mitogenic activityof bFGF was verified by a thymidine incorporation assay involving HUVEcells and various combinations of bFGF, Cyr61, and anti-Cyr61antibodies. Cells were plated and grown as described above. Thefollowing combinations of supplements (final plate concentrations notedparenthetically) were then pre-incubated for 1 hour before addition toindividual plates: 1) pre-immune antiserum (3%); 2) bFGF (15 ng/ml) andpre-immune antiserum (3%); 3) pre-immune antiserum (3%) and Cyr61 (4μg/ml): 4) pre-immune antiserum (3%), Cyr61 (4 μg/ml), and bFGF (15ng/ml): 5) anti-Cyr61 antiserum (3%); 6) anti-Cyr61 antiserum and bFGF(15 ng/ml): 7) anti-Cyr61 antiserum (3%) and Cyr61 (4 μg/ml); and 8)anti-Cyr61 antiserum (3%). Cyr61 (4 μg/ml), and bFGF (15 ng/ml).

[0174] Following incubation in the presence of [³H]-thymidine asdescribed above, cells exposed to pre-immune antiserum incorporatedabout 2×10² cpm thymidine; cells contacting pre-immune antiserum andbFGF incorporated 1.3×10³ cpm; cells receiving pre-immune antiserum andCyr61 incorporated 1×10² cpm; cells receiving pre-immune antiserum.Cyr61, and bFGF incorporated 3.6×10³ cpm; cells exposed to anti-Cyr61antiserum incorporated 2×10² cpm: cells receiving anti-Cyr61 antiserumand bFGF incorporated about 1.3×10³ cpm; cells contacting anti-Cyr61antiserum and Cyr61 incorporated about 1×10² and cells receivinganti-Cyr61 antiserum. Cyr61, and bFGF incorporated 1×10₃ cpm. Theseresults indicate that pre-immune antiserum had no effect onCyr61-induced potentiation of bFGF mitogenic activity. Anti-Cyr61antiserum. however, completely abolished the potentiation of bFGF byCyr61. Moreover, the effect of anti-Cyr61 antiserum was specific toCyr61-induced mitogenic potentiation because anti-Cyr61 antiserum had noeffect on the mitogenic activity of bFGF per se. Therefore, Cyr61 can beused as a reagent to screen for useful mitogens.

[0175] DNA synthesis for HUVE cells and NIH 3T3 fibroblasts was measuredby thymidine incorporation as described previously (Kireeva et al., Mol.Cell. Biol. 16: 1326-1334 [1996]) with minor modifications. HUVE cellswere grown in 24-well plates to a subconfluent state, serum-starved for24 hours and treated with F12K medium containing 10% fetal bovine serum(FBS), 1 μCi/ml [³H]-thymidine and 10 ng/ml basic Fibroblast GrowthFactor (bFGF) (Gibco-BRL, Inc.) with various concentrations of Cyr61 andFisp12 as indicated. NIH 3T3 fibroblasts were grown to subconfluence,serum-starved for 48 hours, and treated with Minimal Essential Medium(MEM) containing 0.5% FBS, 1 μCi/ml [³H]thymidine, bFGF and variousconcentrations of Cyr61 or Fisp12. Thymidine incorporation into thetrichloroacetic acid-insoluble fraction was determined after 24 hourincubation. Logarithmically grown mink lung epithelial cells (Mv1lu.CCL64) were treated with various concentrations of TGF-β1 (Gibco-BRL)and 2 μg/ml of Cyr61 or Fisp12 for 18 hours: [³H]-thymidine was thenadded to 1 μCi/ml for 2 hours. Thymidine incorporation was determined asdescribed above.

[0176] Purified recombinant Fisp12 protein did not exhibit any mitogenicactivity under any tested assay conditions. Rather, Fisp12 was able toenhance DNA synthesis induced by fibroblast growth factor in either NIH3T3 fibroblasts or HUVE-cells. This activity was nearlyindistinguishable from that exhibited by Cyr61.

[0177] Whereas in fibroblasts and endothelial cells, Cyr61 and Fisp12enhance growth factor-induced DNA synthesis, both proteins can alsoenhance growth factor-mediated actions in another way. It is known thatTGF-β acts to inhibit DNA synthesis in epithelial cells (Satterwhite etal., 1994). It was observed that both Cyr61 and Fisp12 enhanced theability of TGF-β to inhibit DNA synthesis in mink lung epithelial cells.The data demonstrate that both recombinant Cyr61 and Fisp12. purifiedfrom serum-free sources. are not mitogenic by themselves, but have theability to synergize with the actions of polypeptide growth factors.Cyr61 and Fisp12 enhance DNA synthesis induction by FGF, and enhance DNAsynthesis inhibition by TGF-β.

[0178] The present invention also comprehends the use of CTGF in methodsto potentiate the mitogenic effect of true growth factors, or to screenfor true growth factors. Those contemplated uses are in contrast to thereported use of CTGF as a mitogen or growth factor itself. U.S. Pat. No.5,408,040, column 7, line 65, to column 11, line 7, incorporated hereinby reference hereinabove.

[0179] Further, the invention comprehends methods of screening formodulators of mitogen potentiation. A comparative assay exposessubconfluent cells to an ECM signalling molecule such as Cyr61, a growthfactor, and a suspected modulator of an ECM signalling molecule. As acontrol, similar cells are exposed to the ECM signalling molecule andthe growth factor. A further control exposes similar cells to the growthfactor and the suspected modulator in the absence of the ECM signallingmolecule. Based on the relative cell proliferation rates, as measuredby, e.g., [³H]-thymidine incorporation. an identification of a suspectedmodulator as a promoter of mitogen potentiation (elevated cellproliferation in the presence of all three molecules) or an inhibitor ofmitogen potentiation (decreased cell proliferation in the presence ofthe three molecules) can be made.

EXAMPLE 19 Cornea Assay For Angiogenic Factors And Modulators

[0180] Another assay for modulators of angiogenesis is an in vivo assayfor assessing the effect of a suspected modulator in the presence of anECM signalling molecule-related biomaterial. such as Cyr61, onangiogenesis is the Cornea Assay. The Cornea Assay takes advantage ofthe absence of blood vessels in the cornea. which in the presence of anangiogenic factor, results in the detectable development of capillariesextending from the sclera into the cornea. Friedlander et al., Science270:1500-1502 (1995). This ingrowth of new blood vessels from the scleracan be microscopically monitored. Further, the visually determined rateof migration can be used to assess changes in the rate of angiogenesis.These cornea assays may be performed using a wide variety of animalmodels. Preferably, the cornea assays are performed using rats. By wayof example. an assay for suspected modulators of Cyr61 using this assayis disclosed. To perform this assay, Cyr61 is initially titrated usingprimary capillary endothelial cells to determine effectiveconcentrations of Cyr61. Subsequently, Cyr61, in the presence or absenceof a suspected modulator, is surgically implanted into the corneas ofmammalian laboratory animals, e.g., rabbits or rats. In a preferredembodiment, Cyr61 (or Cyr61 and a suspected modulator) is embedded in abiocompatible matrix, using matrix materials and techniques that arestandard in the art. Subsequently. eyes containing implants are visuallyobserved for growth of the readily visible blood vessels within the eye.Control implantations may consist of physiologically balanced buffersembedded in the same type of matrix and implanted into eyes of the sametype of laboratory animal receiving the Cyr61-containing implants.

[0181] The development of an in vivo cornea assay for angiogenic factorshas advantages over existing in vitro assays for these factors. Theprocess of angiogenesis involves four distinct phases: induction ofvascular discontinuity, endothelial cell movement, endothelial cellproliferation, and three-dimensional restructuring and sprouting. Invitro assays can evaluate only two of these steps: endothelial cellmigration and mitogenesis. Thus, to provide a comprehensive assay forangiogenic factors, an in vivo assay such as the cornea assay ispreferred.

[0182] The cornea assay has been used to confirm the effect ofangiogenic factors such as Cyr61, Fisp12, CTGF, and Nov, on the processof angiogenesis. Moreover, modifying the cornea assay by including anyof these angiogenic factors and a suspected modulator of their activityresults in a cornea assay for modulators of angiogenesis. For example,in one embodiment of the invention, dose of an angiogenic factor such asCyr61 could be used in cornea assays for positive effectors of theangiogenic activity of Cyr61. An appropriate dose of Cyr61 wouldinitially be determined by titration of the dose response relationshipof Cyr61 with angiogenic events. Inclusion of a control assay lackingCyr61 would eliminate compounds having a direct effect on angiogenesis.In an alternative embodiment of the invention, an effective dose of anangiogenic factor such as Cyr61 could be used to assay for negativemodulators of the activity of an angiogenic factor. In yet anotheralternative embodiment, a corneal implant comprises Cyr61 and anothercorneal implant comprises Cyr61 and a suspected modulator ofangiogenesis. Measurements of the development of blood vessels in theimplanted corneas provides a basis for identifying a suspected modulatoras a promoter of angiogenesis (elevated blood vessel development in thecornea containing an implant comprising the suspected modulator. Arelative decrease in blood vessel development identifies an inhibitor ofangiogenesis.

[0183] The rat is preferred as the animal model for the cornea assay.Disclosures in the art have established the rat model as awell-characterized system for analyzing angiogenesis. Parameters such asimplant size, protein release dynamics, and suitable surgicaltechniques, have been well characterized. Although any strain of rat canbe used in the cornea assay, preferred strains will bewell-characterized laboratory strains such as the Sprague-Dawley strain.

[0184] Although rats of various sizes can be used in the cornea assay. apreferred size for the rats is 150-200 g/animal. Anesthesia is inducedwith Methoxyflurane and is maintained for 40-60 minutes with sodiumpentobarbital (50 mg/kg. delivered intraperitoneally). The eyes aregently opened and secured in place by clamping the upper eyelid with anon-traumatic hemostat. Two drops of sterile proparacaine hydrochloride(0.5%) are then placed on each eye as to effect local anesthesia. Usinga suitable surgical blade such as a No. 11 Bard Parker blade, anapproximately 1.5 mm incision is made approximately 1 mm from the centerof the cornea. The incision extends into the stroma but not through it.A curved iris spatula approximately 1.5 mm in width and approximately 5mm in length is then inserted under the lip of the incision and gentlyblunt-dissected through the stroma toward the outer canthus of the eye.Slight finger pressure against the globe of the eye helps to steady theeye during dissection. The spatula penetrates the stroma no more thanapproximately 2.5 mm. Once the cornea pocket is made, the spatula isremoved and the distance between the limbus and base of the pocket ismeasured to make sure the separation is at least about 1 mm.

[0185] To provide slow release of the protein after implantation in thecornea, protein is mixed with poly-2-hydroxyethylmethacrylate (Hydron),or an equivalent agent, to form a pellet of approximately 5 μl. Implantsmade in this way are rehydrated with a drop of sterile lactated Ringerssolution and implanted as described above. After implantation, thecorneal pocket is sealed with erythromycin ointment. After implantation,the protein-Hydron pellet should remain near the limbus of the cornea(cornea-sclera border) and vision should not be significantly impaired.

[0186] Following surgery, animals are examined daily for seven days withthe aid of a stereomicroscope to check for inflammation and responses.To facilitate examination, the animal is anesthetized withMethoxyflurane and the anesthetic is continuously administered by nosecone during examination. During this seven day period, animals aremonitored for implant position and corneal exudate. Animals exhibitingcorneal exudate are sacrificed. A preferred method of euthanasia isexsanguination. Animals are initially anesthetized with sodiumpentobarbital (50 mg/kg) and then perfused, as described below.

[0187] After seven days. animals are perfused with colloidal carbon(e.g., India Ink). Anesthesia is induced with Methoxyflurane, and ismaintained with sodium pentobarbital (50 mg/kg, intraperitoneally). Eachanimal is perfused with 100-200 ml warm (37° C.) lactated Ringerssolution per 150 g of body mass via the abdominal aorta. Once the snoutof the animal is completely blanched, 20-25 ml of colloidal carbon isinjected in the same way as the Ringers solution, until the head andthoracic organs are completely black. Eyes are then enucleated andfixed. Corneas are excised, flattened, and photographed.

[0188] Each protein is typically tested in three doses, in accordancewith the practice in the art. Those of ordinary skill in the art realizethat six positive corneal responses per dose are required to support anidentification of an angiogenic response. An exemplary cornea assayincludes three doses of the protein under study, with six rats beingtested at each dose. Additionally, six animals are exposed to abuffer-Hydron implant and serve as negative controls. Exposure of atleast three animals to a known angiogenic factor-Hydron implant serve aspositive controls. Finally, to demonstrate the specificity of anyobserved response, six animals are exposed to implants containing asingle dose of the protein under study an excess of neutralizingantibody, and Hydron.

[0189] A cornea assay as described above was performed to assess theability of Cyr61 to induce angiogenesis. Four animals were givennegative control implants containing a buffer-Hydron pellet (both eyes).Each of these animals failed to show any blood vessel development ineither eye after seven days. Six animals received implants containing abiologically effective amount of Fibroblast Growth Factor (0.15 μM) inone eye and a control pellet in the other eye: all six showed angiogenicdevelopment in the eye receiving FGF, none showed neovascularization inthe eye receiving the negative control. Seven animals received 1 μg/mlCyr61 in one eye and all seven of these eyes showed blood vessel growth;one of the seven eyes receiving a negative control showed angiogenicdevelopment. Finally, four animals received implants locally releasing 1μg/ml Cyr61 (Hydron prepared with a 10 μg/ml Cyr61 solution) and aspecific anti-Cyr61 antibody in three-fold excess over Cyr61: none ofthe eyes of this group showed any angiogenic development. Thus, the invivo assay for angiogenesis identifies angiogenic factors such as FGFand Cyr61. The assay also is able to reveal inhibition of angiogenicdevelopment induced ECM signalling molecules such as Cyr61.

EXAMPLE 20 Blood Clotting

[0190] ECM signalling molecules are also useful in correctinghemostasis, or abnormal blood clotting. A defect in blood clottingcaused by, e.g., low level expression of cyr61 which thereby allowsTissue Factor Pathway Inhibitor (TFPI) to act unchecked can be correctedby expression or use of recombinant Cyr61 protein.

[0191] Cyr61 can interact with TFPI, a protein that inhibits extrinsicblood coagulation. TFPI inhibits blood clotting in a two step process.First, TFPI binds to factor Xa and the TFPI:Xa complex then interactswith the Tissue Factor (TF):Factor VIIa complex, thereby inhibiting thelatter complex. The TF:Factor VIIa complex is the complex that activatesfactors IX and X. By inhibiting TF:VIIa. TFPI regulates coagulation bypreventing the activation of Factors IX and X, required for bloodclotting. The interaction of Cyr61 with TFPI inhibits the activity ofTFPI, thus promoting blood coagulation. Cyr61 is, thus, a tissue factoragonist.

[0192] Because of the interaction of Cyr61 and TFPI, Cyr61 can controlthe ability of TFPI to inhibit coagulation, thereby regulatinghemostasis. A defect in Cyr61 may lead to the inability to inhibit TFPIat the appropriate time, resulting in excessive inhibition of tissuefactor, thereby preventing clot formation. Deregulated expression ofCyr61 will conversely inhibit the activity of TFPI constitutively, andthus tissue factor is constantly active, resulting in excessiveclotting. When the expression of Cyr61 in endothelial cells isderegulated, one possible outcome is thrombosis.

[0193] In addition to Cyr61, other ECM signalling molecules, such asFisp12 and CTGF, have been shown to exert effects on cells participatingin angiogenesis. Consequently, it is anticipated that a variety of ECMsignalling molecule-related biomaterials, alone or in combination, maybe used in the methods of the invention directed towards modulatinghemostasis.

EXAMPLE 21 Ex vivo Hematopoietic Stein Cell Cultures

[0194] To investigate the effect of Cyr61 on the growth of primitivemultipotent stem cells, several assays that distinguish these cells frommore mature progenitor cells in a hematopoietic culture are employed.These assays make use of physicochemical (fibronectin-binding) or growthand development-related (generation of progenitor blast colonies)differences between immature and mature subsets of cells.

[0195] Two cell lines which require conditioned media for growth areused as a source of hematopoietic stem cells (HSC). These cloned,factor-dependent murine lines are B6Sut (cloned from long term bonemarrow culture and capable of growing in liquid medium withoutdifferentiation, but multipotent in agar, as described in Greenberger etal, Proc. Natl. Acad. Sci. [USA] 80:2931 [1983]), and FDCP-mix (clonedfrom long term bone marrow culture cells infected with the recombinantvirus src-MoMuLV, and are multipotent in agar cultures, as described inSpooncer et al., Nature 310:2288 [1984]). B6Sut cells are propagated inKincaid's medium with 10% fetal calf serum (FCS) and 10% 6×-concentratedWEHI-conditioned medium. Greenberger et al. FDCP-mix cells arepropagated in Fischer's medium with 20% horse serum and 10%6×-concentrated WEHI-conditioned medium. The cell lines are propagatedat 37° C. 5% CO₂.

[0196] Various ex Vivo or in Vitro cultures are assayed for populationgrowth in the presence or absence of exogenously supplied murine Cyr61or polyclonal anti-Cyr61 antibodies. Under limiting dilution conditions,the cobblestone area forming cell (CAFC) assay is used to identify cellswith long term repopulating ability. Ploemacher et al., Blood 74:2755(1989): Ploemacher et al., Blood 78:2527 (1991). Cells identified ashaving long term repopulating ability by the CAFC assay are thenanalyzed by measuring three parameters: Rate of population doubling,mitotic index, and rate of DNA synthesis.

[0197] Long term cultures, with or without supplementation with Cyr61,are assayed for their levels of primitive HSC in the CAFC assay. van derSluijs et al., Exp. Hematol. 22:1236 (1994). For example, M2-10B4stromal cells, B6Sut, and FDCP-mix are each subjected to the CAFC assayin the following manner, described for the M2-10B4 cell line. Stromalcell layers are prepared by inoculating 5×10⁵ M2-10B4 stromal cells (acell line cloned from bone marrow stroma, Sutherland et al. Blood 78:666[1991]) into each well of a 96-well culture plate in DMEM with 10% FCS.When the cells approach confluency, they are rinsed with PBS andirradiated (20 Gy of gamma-irradiation, 1.02-1.04 Gy/minute) to preventreplication of any hematopoietic cells within the stroma, withoutaffecting the stroma's ability to support hematopoiesis.

[0198] Hematopoietic stem cells are added to the irradiated stromalcells in DMEM with 10% FCS, in the presence or absence of Cyr61 (10μg/ml final concentration). Population doubling rates are determined.e.g., by microscopic examination of cell morphology to determine thenumbers of long term repopulating cells (and more mature short termprogenitor cells) present in the various experimental long termcultures. Subsequent investigation of the expansion and differentiationcapacities of the potential long term HSC cultures is used forconfirmation of suitable candidate cell lines.

[0199] The mitotic index is determined according to procedures standardin the art. Keram et al., Cancer Genet. Cytogenet. 55:235 (1991).Harvested cells are fixed in methanol:acetic acid (3:1, v:v), counted,and resuspended at 10⁶ cells/mil in fixative. Ten microliters of thissuspension is placed on a slide, dried, and treated with Giemsa stain.The cells in metaphase are counted under a light microscope, and themitotic index is calculated by dividing the number of metaphase cells bythe total number of cells on the slide. Statistical analysis ofcomparisons of mitotic indices is performed using the 2-sided pairedt-test.

[0200] The rate of DNA synthesis is measured using a thymidineincorporation assay. Various cultures are propagated in 1 μCi/ml[³H]-thymidine (ICN Biomedicals, Inc., Costa Mesa, Calif.) for 24-72hours. Harvested cells are then rinsed with PBS and fixed with 10%trichloroacetic acid. DNA is dissolved in 0.1 N NaOH, and thymidineincorporation is determined, for example by liquid scintillationspectrophotometry.

[0201] The use of an ECM signalling molecule-related biomaterial. suchas Cyr61 , can be used in the ex vivo expansion of hematopoietic stemcell cultures. In addition, more than one ECM signallingmolecule-related biomaterial may be used to expand these cultures. Forexample. Cyr61, with its expression targeted locally, may be combinedwith Fisp12, which exhibits a more expansive targeting as evidenced bythe presence of Fisp12 in culture media. As an alternative, CTGF may besubstituted for Fisp12, its mouse ortholog. One of skill in the artwould be able to devise other combinations of ECM signallingmolecule-related biomolecules that are within the spirit of theinvention.

[0202] Those of ordinary skill in the art will recognize that thesuccessful expansion of hematopoietic stem cell cultures in the presenceof ECM signalling molecules such as Cyr61 provides a basis for a methodof screening for suspected modulators of that expansion process. As inthe other methods of the invention, a suspected modulator is combinedwith an ECM signalling molecule such as Cyr61 and exposed to primitivecells. In parallel, the ECM signalling molecule is exposed to similarcells. The relative rates of expansion may be used to identify apromoter, or inhibitor, of the ability of the ECM signalling molecule toexpand pluripotent hematopoietic stem cell cultures.

[0203] Cyr61, alone or in combination with other hematopoietic growthfactors, may also be used to expand stem cell populations taken from apatient and which may, after expansion, be returned to the patient orother suitable recipient patient after for example, chemotherapy orother treatment modalities that result in the depletion of blood cellsin a patient. Stem cell populations expanded according to the presentinvention may also be used in bone marrow transplants in a patient inneed thereof.

EXAMPLE 22 Organ regeneration

[0204] The role of Cyr61 in the various cellular processes invoked bychanges in the cellular growth state indicate that this protein would beeffective in promoting organ regeneration. Towards that end, studieswere conducted to determine the expression profile of murine cyr61 inremaining liver tissue following a partial hepatectomy. (The response ofremaining liver tissue following partial hepatectomy is a model for theliver's response to a variety of injuries, including chemical injuries,e.g., exposure to toxic levels of carbon tetrachloride.)

[0205] BALB/c 3T3 (Charles River) mice were subjected to partialhepatectomies removing approximately 67% of their liver tissue. Higginset al., Archs. Path. 12:186-202 (1931). Twenty microgram aliquots of RNAwere removed from the remaining liver tissue at varying times followingthe operation and liver RNA was isolated by tissue homogenizationfollowed by guanidinium isothiocyanate, cesium chloride precipitation.Sambrook et al. RNAs were then immobilized on nitrocellulose filters andprobed with radiolabeled clones containing various regions of murinecyr61 cDNA. Results were visualized by autoradiography and indicatedthat removal of liver tissue induced cyr61 mRNA expression, particularlyin cells found near the injury site. Consequently. induction of cyr61expression, e.g., by recombinant techniques, might promote theregeneration of organs such as liver. For example, Cyr61 expression canbe controlled, e.g., by introducing recombinant cyr6 constructs thathave been engineered to provide the capacity to control expression ofthe gene, e.g., by the use of tissue-specific promoters, e.g., the K14promoter for expression in skin. The recombinant cyr61 may be introducedto cells of the relevant organ by gene therapy techniques using vectorsthat facilitate homologous recombination (e.g., vectors derived fromHerpesviruses, Adenovirus, Adeno-associated Virus, Cytomegalovirus,Baculovirus, retroviruses, Vaccinia Virus, and others). Techniques forintroducing heterologous genes into eukarvotic cells. and techniques forintegrating heterologous genes into host chromosomes by homologousrecombination, are well known in the art.

[0206] The development of skin, another organ, is also affected byCyr61. The expression of Cyr61 is induced in cells in the vicinity ofskin injuries. Also, as described above, Cyr61 has a chemotactic effect(i.e., Cyr6 induces cell migration) on endothelial cells andfibroblasts. Further, Cyr61 induces the proliferation of endothelialcells and fibroblasts. Both processes are involved in the healing ofskin wounds. Accordingly, Cyr61 administration. e.g., by localized ortopical delivery, should promote skin regeneration.

[0207] Cyr61 is also highly expressed in lung epithelium. These cellsare frequently injured by exposure to environmental contaminants. Inparticular, lung epithelium is frequently damaged by air-borne oxidants.The administration of Cyr61. e.g., in atomizers or inhalers, maycontribute to the healing of lung epithelium damaged, e.g., byenvironmental contaminants.

EXAMPLE 23 Chondrogenesis—ECM Signalling Molecules Are Expressed inMesenchyme

[0208] Some ECM signalling molecules are also expressed in cells, suchas Mesenchyme cells, that ultimately become a part of the skeletalsystem. In this Example, Cyr61 is identified as one of the ECMsignalling molecules expressed in mesenchyme cells. Limb mesenchymalcells were grown in micromass culture as described above on glasscoverslips (Fisher) for 3 days. Cultures were fixed in 4%paraformaldehyde in PBS, incubated for 30 minutes at room temperaturewith 1 mg/ml bovine testicular hyaluronidase (type IV, Sigma) in 0.1Nsodium acetate (pH 5.5) with protease inhibitors phenymethylsulfonylfluoride (PMSF, 1 mM), pepstatin (1 μg/ml), leupeptin (1 μg/ml),aprotinin (1 μg/ml), aminocaproic acid (50 mM), benzamidine (5 mM). andEDTA (1 mM), blocked with 10% goat serum in PBS and incubated overnightat 4 C. with primary antibodies against Cyr61 (Yang et al., 1991),fibronectin (Gibco) and tenascin (Gibco). Controls were incubated withanti-Cyr61 antibodies neutralized with 1 μg/ml purified Cyr61. Cultureswere subsequently incubated with FITC-conjugated goat-anti-rabbitsecondary antibody (Zymed), for 1 hour at room temperature.

[0209] For whole mount immunohistochemical staining, mouse embryos fromgestational days 10.5 to 12.5 were fixed in 4% paraformaldehyde in PBS,dehydrated in methanol/PBS and stored at −20 C. in absolute methanol.After rehydration, embryos were incubated with anti-Cyr6 1 antibodies asdescribed in Hogan et al., Development 120:53-60 (1994), incorporatedherein by reference. Controls were incubated with anti-Cyr61 antibodiesneutralized with 1 μg/ml purified Cyr61 . Immunostained embryos werefixed, cleared and photographed.

[0210] Consistent with the transient expression of the Cyr61 mRNA insomitic mesenchymal cells that are differentiating into chondrocytes(O'Brien et al., 1992), the Cyr61 protein was found in the developingembryonic skeletal system. Cyr61 was localized by whole mountimmunohistochemical staining to the proximal limb bud mesenchyme ingestational day 10.5 to 12.5 embryos. The Cyr61 protein was localized tothe developing vertebrae, the calvarial frontal bone and the firstbrachial arch, as well as in the heart and umbilical vessels, forming anexpression pattern that was consistent with the cyr61 mRNA expressionpattern (O'Brien et al., 1992).

[0211] Cyr61 protein could be detected by immunoblot analysis in wholelimb buds and in micromass cultures of limb bud mesenchymal cells. Thelevel of Cyr61 protein remained at relatively constant levels throughoutthe 4 day culture period during which chondrogenesis occurred. Usingquantitative immunoblot analysis, Cyr61 was estimated to representapproximately 0.03% of total cellular and extracellular proteins in themesenchymal cell cultures. Cyr61, tenascin (Gibco), and fibronectin werelocalized to the cartilage nodules by immunofluorescent staining in themesenchymal cell cultures. Cyr61 and tenascin were primarily localizedamong the intranodular cells, while a fibrillar staining pattern wasalso observed around and between the cartilage nodules withanti-fibronectin antibodies. A similar immunofluorescent stainingpattern was observed in transverse sections of the micromass culturesfor all three antibodies. Together, these results show that endogenousCyr61 is localized in the developing limb bud mesenchyme, both in vivoand in vivo.

EXAMPLE 24 Chondrogenesis—ECM Signalling Molecules Promote Cell Adhesion

[0212] Cyr61 is a secreted protein that mediates the adhesion offibroblasts and endothelial cells to non-tissue culture-treated plasticsurfaces (Kireeva et al., Mol. Cell. Biol. 16:1326-1334 [1996]). Theattachment of limb bud mesenchymal cells on non-tissue culture dishescoated with BSA. Cyr61, tenascin, and fibronectin, were compared.

[0213] Cyr61, fibronectin (Gibco), or tenascin (Gibco) were diluted in0.1% protease-free bovine serum albumin (BSA) in PBS with 0.5 mM PMSF,to a final concentrations of 10 or 50 μg/ml. A 10 μl drop/well wasplaced in a non-tissue culture treated 24-well plate (Corning), andincubated at room temperature for 2 hours. The wells were blocked with1% BSA in PBS for 1 hour at room temperature. and rinsed with serum-freeMEM (Modified Eagle's Medium). Limb mesenchymal cells, suspended at5×10⁵ cell/ml in serum-free MEM, were added at a volume of 400 μl/well,and incubated at 37 C., 5% CO₂ for 1 or 3 hours. At each time point, thecell suspension was removed, the wells were rinsed with MEM and theremaining adherent cells were photographed.

[0214] Cells attached poorly to BSA-coated dishes, but adhered asclusters of rounded cells to Cyr61- and tenascin-coated dishes within 1hour of plating. In contrast, cells plated on fibronectin-coated dishesattached uniformly and started to spread. When cells were allowed toattach for 3 hours. many more adherent cells were observed. Furthermore,intercellular clustering and rounded cell morphology were maintained incells plated on Cyr61 and tenascin, while cells plated on fibronectinspread to form a monolayer. These observations show that Cyr61 mediatesthe adhesion and maintenance of a rounded cellular morphology which isconducive for mesenchymal cell chondrogenesis (Zanetti et al., Dev.Biol. 139:383-395 [1990]: Solursh et al., Dev. BIol. 94:259-264 [1982]),similar to that previously reported for tenascin (Mackie et al., J. CellBiol. 105:2569-2579 [1987]).

[0215] As mentioned previously, ECM signalling molecules such as Cyr61may be used in methods of screening for modulators of cell adhesionincluding, but not limited to, the adhesion of chondrocytes. Thecomparative assay, described above, measures the relative adhesionlevels of cells exposed to a combination of an ECM signalling moleculeand a suspected modulator of cell adhesion and cells exposed to the ECMsignalling molecule alone. whereby the relative levels provide a basisfor identifying either a promoter or an inhibitor of cell adhesion.

EXAMPLE 25 Chondrogenesis—ECM Signalling Molecules Promote CellAggregation

[0216] Since aggregation is an essential step for chondrogenicdifferentiation (Solursh, M., In The role of extracellular matrix indevelopment, pp. 277-303 (Trelstad, R., ed.) (Alan R. Liss, N.Y. 1984)),the ability of Cyr61 to mediate intercellular aggregation in suspensioncultures of mesenchymal cells was assessed. The number of cellsremaining at various times after isolation were counted. Untreatedmesenchymal cells in suspension began to aggregate soon after isolation,as the number of single cells was decreased to 30% of the initial numberwithin a 2 hour incubation period. Cell aggregation was significantlyinhibited in cultures treated with affinity-purified anti-Cyr61antibodies, indicating that endogenous Cyr61 is important formesenchymal cell aggregation. To rule out the possibility that theaffinity-purified anti-Cyr61 antibodies might contain undefinedcomponents that interfere with aggregation, anti-Cyr61 antibodies,described above. were pre-incubated with purified Cyr61 protein prior toaddition to cells. These pre-incubated antibodies affected cellaggregation no more than the IgG and Cyr61 buffer controls, indicatingthat the anti-Cyr61 antibodies achieved their inhibition of cellaggregation by neutralizing the endogenous Cyr61 protein of mesenchymalcells.

[0217] In addition to the cell aggregation in suspension culturesdescribed above, the effect of Cyr61 on mesenchymal cell aggregation inmicromass cultures was also examined. When purified Cyr61 protein (0.3μg/ml) was added to limb mesenchymal cells, precocious cellularaggregation was observed within 24 hours, unlike control cells which hadnot received Cyr61. Neither Cyr61-treated nor control cultures haddifferentiated into cartilage nodules at this time. By culture day 3,the development of internodular cellular condensations between thedistinct cartilage nodules was more extensive in Cyr61-treated cultures.These internodular condensations subsequently undergo chondrogenesis,observed as Alcian blue-staining cartilaginous matrix on culture day 4.Taken together, these results indicate that Cyr61 is able to promotecell-cell aggregation, a necessary step in chondrogenesis of mesenchymalcells in micromass culture.

EXAMPLE 26 Chondrogenesis—ECM Signalling Molecules Promote CellProliferation

[0218] At Some ECM signalling molecules, such as Cyr61, affectchondrogenesis, as revealed by effects on limb bud mesenchyme cells inmicromass culture, as described above. Ahrens et al., Dev. Biol.60:69-82 (1977), has reported that these cells, in micromass culture,undergo chondrogenesis in a manner similar to the in vivo process.Mesenchyme cells were obtained from mouse embryonic limb buds by trypsindigestion (1 mg/ml. 1:250 dilution of porcine pancreatic trypsin, SigmaChemical Co.). Cells were explanted in plastic tissue culture wells andallowed to attach for 2 hours at 37° C., 5% CO₂. Cells were thenincubated for 24 hours at 37° C. 5% CO₂ in MEM with 10% FBS, penicillin(50 U/ml), and streptomycin (50 μg/ml). At this point, the compositionof the medium was changed by substituting 4% NuSerum (CollaborativeBiomedical Products, Inc.) for 10% FBS. Individual cultures thenreceived Cyr61, fibronectin. heparin, (each at approximately 1 μg/ml) orbuffer as a negative control. An additional control was provided byadding a 1:100 dilution of affinity-purified anti-Cyr61 antibody(approximately 13 μg/ml stock solution), elicited and purified bystandard techniques. Harlow et al.

[0219] Cell proliferation was assessed by the thymidine assay, describedabove, and by microscopic cell counts. Chondrogenesis was assessed byquantifying the incorporation of [³⁵S]-sulfate (ICN Biomedicals, Inc.)into sulfated glycosaminoglycans, and by qualitatively determining theextent of chondrogenesis by cell staining with Alcian Blue. Cultures,described above, were labeled with 2.5 μCi/ml [³⁵S]-sulfate for 18hours, washed twice in PBS. fixed with Kahle's fixative (Pepper et al.,J. Cell Sci. 109:73-83 [1995]) and stained for 18 hours in 0.5% AlcianBlue, pH 1.0. The extent of chondrogenesis is correlated with theintensity of Alcian Blue staining. San Antonio et al., Del. Biol.115:313-324 (1986). The results show that Cyr61 specifically increasedlimb bud mesenchyme cell proliferation and aggregation, leading toenhanced chondrogenesis.

[0220] In addition to demonstrating that purified Cyr61 enhanced growthfactor-induced DNA synthesis in fibroblasts and endothelial cells, theeffects of Cyr61 on cell proliferation were directly examined. Cellproliferation during the 4 day culture period was determined by countingcell number and by incorporation of [³H]-thymidine. To determine cellnumber, cells were harvested by trypsin/EDTA (Sigma) and counted with aCoulter counter. In parallel cultures, [³H]-thymidine (1 μCi/ml; ICN)was added to the media for 18 hours and incorporation in theTCA-insoluble layer was determined by liquid scintillation counting.Purified Cyr61 protein added to limb mesenchymal cells both increasedcell number and enhanced DNA synthesis after 2 and 3 days in culture,although the total cell number in Cyr61-treated and Cyr61-untreatedcultures leveled off at the same level after 4 days.

[0221] The role of Cyr61 in chondrogenesis may also improve theintegration of prosthetic devices. For example, skeletal injuries andconditions frequently are treated by the introduction of a prosthesise.g., hip prosthesis, knee prosthesis. Beyond questions ofhistocompatibility, the successful implantation of a prosthetic devicerequires that the foreign element become integrated into the organism'sskeletal structure. The capacity of Cyr61 polypeptides to affect celladhesion, migration, and proliferation, and the ability of Cyr61polypeptides to induce the differentiation of mesenchyme cells intochondrocytes, should prove valuable in the treatment of skeletaldisorders by prosthesis implantation. For example. integration of aprosthetic device by chondrocyte colonization would be promoted bytherapeutic treatments involving the administration of Cyr61 in apharmaceutically acceptable adjuvant, carrier or diluent, using any ofthe administration routes known in the art or by coating the prosthesisdevice with Cyr61 polypeptides in a suitable carrier. The carrier mayalso be a slow-release type vehicle to allow sustained release of thepolypeptides.

[0222] As noted in previously, the methods of the invention include amethod of screening for modulators of cell proliferation, includingchondrocytes. A comparison of the relative rates of cell proliferationin the presence of a control comprising an ECM signalling molecule alone(e.g., Cyr61) and in the presence of a combination of an ECM signallingmolecule and a suspected modulator of cell proliferation provides abasis for identifying a suspected modulator as a promoter, or inhibitor,of chondrocyte proliferation.

EXAMPLE 27 Chondrogenesis—ECM Signalling Molecules PromoteChondrogenesis

[0223] Chondrogenic differentiation was quantitated by incorporation of[³S]-sulfate (ICN) into sulfated glycosaminoglycans and assessedqualitatively by Alcian Blue staining. Cultures were radiolabeled with2.5 μCi/ml [³S]-sulfate for 18 hr, fixed with Kahle's fixative andstained with 0.5% Alcian Blue, pH 1.0 (Lev et al., 1964). The extent ofchondrogenesis is correlated with the intensity of Alcian Blue staining(San Antonio et al. 1986). [³⁵S]-Sulfate incorporation in the fixed celllayer was quantitated by liquid scintillation counting.

[0224] Exogenous purified Cyr61 protein promoted limb mesenchymal cellaggregation and resulted in greater Alcian blue-staining cartilaginousregions in micromass cultures, suggestive of a chondrogenesis-promotingeffect. This effect was quantified by the incorporation of [³⁵S]-sulfateinto sulfated glycosaminoglycans (San Antonio et al., 1986) inCyr61-treated micromass cultures. Exogenous Cyr61 enhanced [³⁵S]-sulfateincorporation in a dose-dependent manner, resulting in a 1.5-fold and3.5-fold increase with 0.3 and 5 μg/ml Cyr61, respectively, and wascorrelated qualitatively by increased Alcian Blue staining. The increaseobserved at the 5 μg/ml Cyr61 dose (120 nM) is an under-estimation ofthe actual extent of chondrogenesis, since some of the large cartilagenodules which were formed at this dose detached from the dish. Culturestreated with 10 μg/ml Cyr61 formed a more massive mound of cartilage.

[0225] A review of the literature indicated that chondrogenesis in limbmesenchymal cell micromass cultures was increased 2-fold with theaddition of 10 μg/ml heparin (San Antonio et al., 1987; Resh et al.,1985) and 3-fold with 50 μg/ml tenascin (200 nM) (Mackie et al., 1987).The results demonstrated that purified Cyr61 was effective atconcentrations (10-100 nM) similar to or less than those of othermolecules known to promote chondrogenesis in this cell system.

[0226] Since a certain threshold cell density must be reached forinitial aggregation to occur (Umansky. 1966; Ahrens et al. 1977).embryonic mesenchymal cells plated at low densities are normally unableto differentiate into chondrocytes, although the addition of exogenousfactors such as heparin or poly-L-lysine (San Antonio et al., 1986; SanAntonio et al., 1987) have been shown to support chondrogenesis in cellsplated under these conditions. Therefore, the ability of Cyr61 topromote differentiation of mesenchymal cells plated at densities aboveand below the threshold for chondrogenesis was assessed. Cells plated at2.5×10⁶ cell/ml incorporated little [³⁵S]-sulfate. However, when Cyr61was added, these sub-threshold density cultures formed nodules andincorporated sulfate to a level similar to that in cultures plated at3×10⁶ cells/mil, which supports chondrogenesis. Therefore. Cyr61 canpromote chondrogenesis in mesenchymal cells plated at non-chondrogenic.sub-threshold densities.

[0227] It is conceivable that when mesenchymal cells are plated in a hhigh density micromass, the extent of chondrogenesis may be maximal andcannot be enhanced further by exogenous factors, which also may not beaccessible to all cells. However, addition of exogenous Cyr61 resultedin a 2-fold enhancement in [³⁵S]-sulfate incorporation in culturesplated at densities ranging from 3 to 10×10⁶ cell/ml. Therefore, Cyr61can further enhance chondrogenesis in high density micromass cultures,which have apparently not reached a maximal degree of differentiation.

[0228] It is possible that the increased [³⁵S]-sulfate incorporation inCyr61-treated cultures is at least partly due to an increase in cellnumber, since Cyr61 also promotes cell proliferation. If this were true,then normalization of sulfate incorporation with respect to cell numberwould eliminate any differences between control and Cyr61-treatedcultures. This was not found to be the case. Cyr61-treated culturesstill showed an approximately 2-fold increase in normalized sulfateincorporation over control, indicating that Cyr61 promotes a netincrease in chondrogenesis. On culture day 2, the sulfate/cell numberratio was lower in Cyr61-treated cultures compared to controls and isreflective of a low level of [³⁵S]-sulfate incorporation relative tocell number, since mesenchymal cells are mostly proliferating ratherthan differentiating in these early stage cultures (Ede. 1983).

[0229] The presence of endogenous Cyr61 in these cells, both in vivo andin vitro, indicates that Cyr61 may indeed function biologically toregulate chondrogenic differentiation. The ability of exogenously addedpurified Cyr61 to promote intercellular aggregation and to increase[³⁵S]-sulfate incorporation and Alcian-blue staining in limb mesenchymalcells demonstrates that Cyr61 can act as a chondrogenesis enhancingfactor. As shown above in Example 11, anti-Cyr61 antibodies canneutralize both the cell adhesion and DNA-synthesis enhancementactivities of Cyr61. Anti-Cyr61 antibodies were added to the mesenchymalcell culture media or mixed the cell suspension prior to plating.Chondrogenesis was inhibited in the cultures treated with anti-Cyr61antibodies, as demonstrated by decreases of [³⁵S]-sulfate incorporationto 50% and 30% of controls, when antibodies were added to the media, andmixed with the cells, respectively. These observations were correlatedwith decreased Alcian Blue staining. However, mixing of the anti-Cyr61antibodies with mesenchymal cells prior to plating resulted in completedetachment in some of the treated cultures within 24 hours.

[0230] To eliminate the possibility of an unidentified component in theantibody preparation as a cause of cell detachment, anti-Cyr61 antibodywas preincubated with 1 μg/ml purified Cyr61 protein prior to mixingwith cells. The inhibition of chondrogenesis in mesenchymal cells mixedwith neutralized anti-Cyr61 antibodies was abolished.

[0231] Generally, the invention contemplates a method of screening formodulators of chondrogenesis. A comparative assay involves the exposureof chondrocytes to either (a) a combination of a suspected modulator ofchondrogenesis and an ECM signalling molecule such as Cyr61, or (b) theECM signalling molecule alone. Measurements of the relative rates ofchondrogenesis then provide a basis for identifying the suspectedmodulator of chondrogenesis as a promoter or inhibitor of that process.

[0232] The results described in this Example demonstrate that endogenousCyr61 is present in mesenchymal cells and is important for theirchondrogenesis. Accordingly, the use of an ECM signalling molecule. suchas Cyr61, to induce bone healing is contemplated by the invention. Forexample, a biologically effective amount of Cyr61 is introduced into amatrix such as a sponge, as described above, and this material is thenapplied to set bone fractures or used to gather together the fragmentsof a comminuted bone fracture. A biodegradable matrix may be employed,or the matrix may be removed at an appropriate later time.Alternatively, Cyr61 may be applied directly to bone. In addition, Cyr61may be applied to inanimate objects such as biocompatible prosthesis, asdescribed in Example 26.

EXAMPLE 28 Genetics

[0233] Another way to control the effects of an ECM signallingmolecule-related biomaterial is to inactivate it by creating dominantnegative mutations in the relevant gene in actively growing and dividingcells. One approach involves the use of recombinant techniques, e.g., tocreate homozygous mutant genotypes in ex vivo cultures such as HSCcultures. Introduction of these cells into an organism, e.g., a humanpatient, would then provide an opportunity for the introduced mutantcells to expand and alter the expression of the ECM signalling moleculein vivo. Mutants homozygous for such a mutation could affect theexpression of an endogenous wild type ECM signalling molecule in othercells. Heterozygous mutants might produce altered ECM signallingmolecules capable of interacting with the wild type ECM signallingmolecule. also being expressed, in such a way that the ECM signallingmolecule's activities are modulated or abolished.

[0234] Furthermore, because of the role played by ECM signallingmolecules such as Cyr61 in regulating chondrogenesis (i.e., skeletaldevelopment), genetic manipulations that alter the expression of humanCyr61 may prove medically important for prenatal screening methods andgene therapy treatments related to skeletal conditions, in addition toangiogenic conditions. For example, the cyr61 gene is expressed whenmesenchymal cells of both ectodermal and mesodermal originsdifferentiate to form chondrocytes. Thus, one of the roles that Cyr61might play is to regulate the commitment of mesenchyme cells tochondrocyte cell lineages involved in skeletal development. Consistentwith this view, transgenic mice overexpressing cyr61 ectopically areborn with skeletal abnormalities. In all cases examined, the presence ofthe skeletal deformities correlates with expression of the transgene.These results suggest that the human form of Cyr61 may also regulatechondrogenesis and skeletal development. It is also possible that thehuman cyr61 gene may correspond to a genetic locus already known toaffect skeletal development or birth defects relating to bonemorphogenesis. Knowledge of the human Cyr61 protein sequence, presentedin SEQ ID NO: 4 herein, and the coding sequence of the cDNA, presentedin SEQ ID NO: 3 herein, provide the basis for the design of a variety ofgene therapy approaches.

[0235] This information also provides a basis for the design of probesuseful in genotypic analyses. e.g., Restriction Fragment LengthPolymorphism analyses. Such analyses are useful in the fields of geneticcounselling, e.g., in diagnosing diseases and conditions and thelikelihood of their occurrence, as well as in forensic analyses.

[0236] By way of example. the materials of the present invention areuseful in the prenatal screening for a variety of conditions ordisorders, including blood disorders, skeletal abnormalities, andcancerous conditions. Well known techniques for obtaining fetal cells,e.g., amniocentesis, provide the materials needed for diagnosis. In oneembodiment of the invention, the fetal cells are expanded and DNA isisolated. In another embodiment, fetal cells are lysed and polymerasechain reactions are performed using oligonucleotide primers according tothe invention. Using either approach. the DNA is then subjected toanalysis. One analytical approach involves nucleotide sequencedetermination of particular regions of cyr61 or of the entire gene. Theavailable human Cyr61 coding sequence, presented in SEQ ID NO: 3 herein,facilitates the design of sequencing primers that brings nucleotidesequence analysis into the realm of practical reality. An alternative tonucleotide sequence analysis is an investigation of the expressioncharacteristics of the fetal nucleic acid. The capacity of the fetalnucleic acid to be expressed might be dispositive in the diagnosis ofCyr61-related angiogenic, chondrogenic, or oncogenic disorders.

[0237] The invention also comprehends a kit comprising Cyr61. The kitsaccording to the invention provide Cyr61 in a form that is useful forperforming the aforementioned methods of the invention. Kits accordingto the invention contain isolated and purified recombinant human Cyr61in a suitable buffer, optionally stabilized by the addition of glycerolfor storage at −20° C. In addition to the Cyr61 provided in the kit, theinvention also contemplates the inclusion of any one of a variety ofbuffering agents, salts of various types and concentrations, andadditional protein stabilizing agents such as DTT, all of which are wellknown in the art. Other kits according to the invention incorporateisolated and purified murine Cyr61. Kits incorporating a Cyr61polypeptide and an inhibitory peptide or an anti-Cyr61 antibody, asdescribed above, are also contemplated.

1 17 1480 base pairs nucleic acid single linear protein CDS 180..1316misc_feature “Mouse cyr61 cDNA coding sequence” 1 CGAGAGCGCC CCAGAGAAGCGCCTGCAATC TCTGCGCCTC CTCCGCCAGC ACCTCGAGAG 60 AAGGACACCC GCCGCCTCGGCCCTCGCCTC ACCGCACTCC GGGCGCATTT GATCCCGCTG 120 CTCGCCGGCT TGTTGGTTCTGTGTCGCCGC GCTCGCCCCG GTTCCTCCTG CGCGCCACA 179 ATG AGC TCC AGC ACC TTCAGG ACG CTC GCT GTC GCC GTC ACC CTT CTC 227 Met Ser Ser Ser Thr Phe ArgThr Leu Ala Val Ala Val Thr Leu Leu 1 5 10 15 CAC TTG ACC AGA CTG GCGCTC TCC ACC TGC CCC GCC GCC TGC CAC TGC 275 His Leu Thr Arg Leu Ala LeuSer Thr Cys Pro Ala Ala Cys His Cys 20 25 30 CCT CTG GAG GCA CCC AAG TGCGCC CCG GGA GTC GGG TTG GTC CGG GAC 323 Pro Leu Glu Ala Pro Lys Cys AlaPro Gly Val Gly Leu Val Arg Asp 35 40 45 GGC TGC GGC TGC TGT AAG GTC TGCGCT AAA CAA CTC AAC GAG GAC TGC 371 Gly Cys Gly Cys Cys Lys Val Cys AlaLys Gln Leu Asn Glu Asp Cys 50 55 60 AGC AAA ACT CAG CCC TGC GAC CAC ACCAAG GGG TTG GAA TGC AAT TTC 419 Ser Lys Thr Gln Pro Cys Asp His Thr LysGly Leu Glu Cys Asn Phe 65 70 75 80 GGC GCC AGC TCC ACC GCT CTG AAA GGGATC TGC AGA GCT CAG TCA GAA 467 Gly Ala Ser Ser Thr Ala Leu Lys Gly IleCys Arg Ala Gln Ser Glu 85 90 95 GGC AGA CCC TGT GAA TAT AAC TCC AGA ATCTAC CAA AAC GGG GAA AGC 515 Gly Arg Pro Cys Glu Tyr Asn Ser Arg Ile TyrGln Asn Gly Glu Ser 100 105 110 TTC CAG CCC AAC TGT AAA CAC CAG TGC ACATGT ATT GAT GGC GCC GTG 563 Phe Gln Pro Asn Cys Lys His Gln Cys Thr CysIle Asp Gly Ala Val 115 120 125 GGC TGC ATT CCT CTG TGT CCC CAA GAA CTGTCT CTC CCC AAT CTG GGC 611 Gly Cys Ile Pro Leu Cys Pro Gln Glu Leu SerLeu Pro Asn Leu Gly 130 135 140 TGT CCC AAC CCC CGG CTG GTG AAA GTC AGCGGG CAG TGC TGT GAA GAG 659 Cys Pro Asn Pro Arg Leu Val Lys Val Ser GlyGln Cys Cys Glu Glu 145 150 155 160 TGG GTT TGT GAT GAA GAC AGC ATT AAGGAC TCC CTG GAC GAC CAG GAT 707 Trp Val Cys Asp Glu Asp Ser Ile Lys AspSer Leu Asp Asp Gln Asp 165 170 175 GAC CTC CTC GGA CTC GAT GCC TCG GAGGTG GAG TTA ACG AGA AAC AAT 755 Asp Leu Leu Gly Leu Asp Ala Ser Glu ValGlu Leu Thr Arg Asn Asn 180 185 190 GAG TTA ATC GCA ATT GGA AAA GGC AGCTCA CTG AAG AGG CTT CCT GTC 803 Glu Leu Ile Ala Ile Gly Lys Gly Ser SerLeu Lys Arg Leu Pro Val 195 200 205 TTT GGC ACC GAA CCG CGA GTT CTT TTCAAC CCT CTG CAC GCC CAT GGC 851 Phe Gly Thr Glu Pro Arg Val Leu Phe AsnPro Leu His Ala His Gly 210 215 220 CAG AAA TGC ATC GTT CAG ACC ACG TCTTGG TCC CAG TGC TCC AAG AGC 899 Gln Lys Cys Ile Val Gln Thr Thr Ser TrpSer Gln Cys Ser Lys Ser 225 230 235 240 TGC GGA ACT GGC ATC TCC ACA CGAGTT ACC AAT GAC AAC CCA GAG TGC 947 Cys Gly Thr Gly Ile Ser Thr Arg ValThr Asn Asp Asn Pro Glu Cys 245 250 255 CGC CTG GTG AAA GAG ACC CGG ATCTGT GAA GTG CGT CCT TGT GGA CAA 995 Arg Leu Val Lys Glu Thr Arg Ile CysGlu Val Arg Pro Cys Gly Gln 260 265 270 CCA GTG TAC AGC AGC CTA AAA AAGGGC AAG AAA TGC AGC AAG ACC AAG 1043 Pro Val Tyr Ser Ser Leu Lys Lys GlyLys Lys Cys Ser Lys Thr Lys 275 280 285 AAA TCC CCA GAA CCA GTC AGA TTTACT TAT GCA GGA TGC TCC AGT GTC 1091 Lys Ser Pro Glu Pro Val Arg Phe ThrTyr Ala Gly Cys Ser Ser Val 290 295 300 AAG AAA TAC CGG CCC AAA TAC TGCGGC TCC TGC GTA GAT GGC CGG TGC 1139 Lys Lys Tyr Arg Pro Lys Tyr Cys GlySer Cys Val Asp Gly Arg Cys 305 310 315 320 TGC ACA CCT CTG CAG ACC AGAACT GTG AAG ATG CGG TTC CGA TGC GAA 1187 Cys Thr Pro Leu Gln Thr Arg ThrVal Lys Met Arg Phe Arg Cys Glu 325 330 335 GAT GGA GAG ATG TTT TCC AAGAAT GTC ATG ATG ATC CAG TCC TGC AAA 1235 Asp Gly Glu Met Phe Ser Lys AsnVal Met Met Ile Gln Ser Cys Lys 340 345 350 TGT AAC TAC AAC TGC CCG CATCCC AAC GAG GCA TCG TTC CGA CTG TAC 1283 Cys Asn Tyr Asn Cys Pro His ProAsn Glu Ala Ser Phe Arg Leu Tyr 355 360 365 AGC CTA TTC AAT GAC ATC CACAAG TTC AGG GAC TAAGTGCCTC CAGGGTTC 1336 Ser Leu Phe Asn Asp Ile His LysPhe Arg Asp 370 375 AGTGTGGGCT GGACAGAGGA GAAGCGCAAG CATCATGGAGACGTGGGTGG GCGGAGGA 1396 AATGGTGCCT TGCTCATTCT TGAGTAGCAT TAGGGTATTTCAAAACTGCC AAGGGGCT 1456 TGTGGACGGA CAGCAGCGCA GCCG 1480 379 amino acidsamino acid linear protein misc_feature “Mouse Cyr61 amino acid sequence”2 Met Ser Ser Ser Thr Phe Arg Thr Leu Ala Val Ala Val Thr Leu Leu 1 5 1015 His Leu Thr Arg Leu Ala Leu Ser Thr Cys Pro Ala Ala Cys His Cys 20 2530 Pro Leu Glu Ala Pro Lys Cys Ala Pro Gly Val Gly Leu Val Arg Asp 35 4045 Gly Cys Gly Cys Cys Lys Val Cys Ala Lys Gln Leu Asn Glu Asp Cys 50 5560 Ser Lys Thr Gln Pro Cys Asp His Thr Lys Gly Leu Glu Cys Asn Phe 65 7075 80 Gly Ala Ser Ser Thr Ala Leu Lys Gly Ile Cys Arg Ala Gln Ser Glu 8590 95 Gly Arg Pro Cys Glu Tyr Asn Ser Arg Ile Tyr Gln Asn Gly Glu Ser100 105 110 Phe Gln Pro Asn Cys Lys His Gln Cys Thr Cys Ile Asp Gly AlaVal 115 120 125 Gly Cys Ile Pro Leu Cys Pro Gln Glu Leu Ser Leu Pro AsnLeu Gly 130 135 140 Cys Pro Asn Pro Arg Leu Val Lys Val Ser Gly Gln CysCys Glu Glu 145 150 155 160 Trp Val Cys Asp Glu Asp Ser Ile Lys Asp SerLeu Asp Asp Gln Asp 165 170 175 Asp Leu Leu Gly Leu Asp Ala Ser Glu ValGlu Leu Thr Arg Asn Asn 180 185 190 Glu Leu Ile Ala Ile Gly Lys Gly SerSer Leu Lys Arg Leu Pro Val 195 200 205 Phe Gly Thr Glu Pro Arg Val LeuPhe Asn Pro Leu His Ala His Gly 210 215 220 Gln Lys Cys Ile Val Gln ThrThr Ser Trp Ser Gln Cys Ser Lys Ser 225 230 235 240 Cys Gly Thr Gly IleSer Thr Arg Val Thr Asn Asp Asn Pro Glu Cys 245 250 255 Arg Leu Val LysGlu Thr Arg Ile Cys Glu Val Arg Pro Cys Gly Gln 260 265 270 Pro Val TyrSer Ser Leu Lys Lys Gly Lys Lys Cys Ser Lys Thr Lys 275 280 285 Lys SerPro Glu Pro Val Arg Phe Thr Tyr Ala Gly Cys Ser Ser Val 290 295 300 LysLys Tyr Arg Pro Lys Tyr Cys Gly Ser Cys Val Asp Gly Arg Cys 305 310 315320 Cys Thr Pro Leu Gln Thr Arg Thr Val Lys Met Arg Phe Arg Cys Glu 325330 335 Asp Gly Glu Met Phe Ser Lys Asn Val Met Met Ile Gln Ser Cys Lys340 345 350 Cys Asn Tyr Asn Cys Pro His Pro Asn Glu Ala Ser Phe Arg LeuTyr 355 360 365 Ser Leu Phe Asn Asp Ile His Lys Phe Arg Asp 370 375 1418base pairs nucleic acid single linear protein CDS 124..1266 misc_feature“Human cyr61 cDNA coding sequence” 3 GGGCGGGCCC ACCGCGACAC CGCGCCGCCACCCCGACCCC GCTGCGCACG GCCTGTCCGC 60 TGCACACCAG CTTGTTGGCG TCTTCGTCGCCGCGCTCGCC CCGGGCTACT CCTGCGCGCC 120 ACA ATG AGC TCC CGC ATC GCC AGG GCGCTC GCC TTA GTC GTC ACC CTT 168 Met Ser Ser Arg Ile Ala Arg Ala Leu AlaLeu Val Val Thr Leu 1 5 10 15 CTC CAC TTG ACC AGG CTG GCG CTC TCC ACCTGC CCC GCT GCC TGC CAC 216 Leu His Leu Thr Arg Leu Ala Leu Ser Thr CysPro Ala Ala Cys His 20 25 30 TGC CCC CTG GAG GCG CCC AAG TGC GCG CCG GGAGTC GGG CTG GTC CGG 264 Cys Pro Leu Glu Ala Pro Lys Cys Ala Pro Gly ValGly Leu Val Arg 35 40 45 GAC GGC TGC GGC TGC TGT AAG GTC TGC GCC AAG CAGCTC AAC GAG GAC 312 Asp Gly Cys Gly Cys Cys Lys Val Cys Ala Lys Gln LeuAsn Glu Asp 50 55 60 TGC AGC AAA ACG CAG CCC TGC GAC CAC ACC AAG GGG CTGGAA TGC AAC 360 Cys Ser Lys Thr Gln Pro Cys Asp His Thr Lys Gly Leu GluCys Asn 65 70 75 TTC GGC GCC AGC TCC ACC GCT CTG AAG GGG ATC TGC AGA GCTCAG TCA 408 Phe Gly Ala Ser Ser Thr Ala Leu Lys Gly Ile Cys Arg Ala GlnSer 80 85 90 95 GAG GGC AGA CCC TGT GAA TAT AAC TCC AGA ATC TAC CAA AACGGG GAA 456 Glu Gly Arg Pro Cys Glu Tyr Asn Ser Arg Ile Tyr Gln Asn GlyGlu 100 105 110 AGT TTC CAG CCC AAC TGT CAA CAT CAG TGC ACA TGT ATT GATGGC GCC 504 Ser Phe Gln Pro Asn Cys Gln His Gln Cys Thr Cys Ile Asp GlyAla 115 120 125 GTG GGC TGC ATT CCT CTG TGT CCC CAA GAA CTA TCT CTC CCCAAC TTG 552 Val Gly Cys Ile Pro Leu Cys Pro Gln Glu Leu Ser Leu Pro AsnLeu 130 135 140 GGC TGT CCC AAC CCT CGG CTG GTC AAA GTT ACC GGG CAG TGCTGC GAG 600 Gly Cys Pro Asn Pro Arg Leu Val Lys Val Thr Gly Gln Cys CysGlu 145 150 155 GAG TGG GTC TGT GAC GAG GAT AGT ATC AAG GAC CCC ATG GAGGAC CAG 648 Glu Trp Val Cys Asp Glu Asp Ser Ile Lys Asp Pro Met Glu AspGln 160 165 170 175 GAC GGC CTC CTT GGC AAG GAG CTG GGA TTC GAT GCC TCCGAG GTG GAG 696 Asp Gly Leu Leu Gly Lys Glu Leu Gly Phe Asp Ala Ser GluVal Glu 180 185 190 TTG ACG AGA AAC AAT GAA TTG ATT GCA GTT GGA AAA GGCAGA TCA CTG 744 Leu Thr Arg Asn Asn Glu Leu Ile Ala Val Gly Lys Gly ArgSer Leu 195 200 205 AAG CGG CTC CCT GTT TTT GGA ATG GAG CCT CGC ATC CTATAC AAC CCT 792 Lys Arg Leu Pro Val Phe Gly Met Glu Pro Arg Ile Leu TyrAsn Pro 210 215 220 TTA CAA GGC CAG AAA TGT ATT GTT CAA ACA ACT TCA TGGTCC CAG TGC 840 Leu Gln Gly Gln Lys Cys Ile Val Gln Thr Thr Ser Trp SerGln Cys 225 230 235 TCA AAG ACC TGT GGA ACT GGT ATC TCC ACA CGA GTT ACCAAT GAC AAC 888 Ser Lys Thr Cys Gly Thr Gly Ile Ser Thr Arg Val Thr AsnAsp Asn 240 245 250 255 CCT GAG TGC CGC CTT GTG AAA GAA ACC CGG ATT TGTGAG GTG CGG CCT 936 Pro Glu Cys Arg Leu Val Lys Glu Thr Arg Ile Cys GluVal Arg Pro 260 265 270 TGT GGA CAG CCA GTG TAC AGC AGC CTG AAA AAG GGCAAG AAA TGC AGC 984 Cys Gly Gln Pro Val Tyr Ser Ser Leu Lys Lys Gly LysLys Cys Ser 275 280 285 AAG ACC AAG AAA TCC CCC GAA CCA GTC AGG TTT ACTTAC GCT GGA TGT 1032 Lys Thr Lys Lys Ser Pro Glu Pro Val Arg Phe Thr TyrAla Gly Cys 290 295 300 TTG AGT GTG AAG AAA TAC CGG CCC AAG TAC TGC GGTTCC TGC GTG GAC 1080 Leu Ser Val Lys Lys Tyr Arg Pro Lys Tyr Cys Gly SerCys Val Asp 305 310 315 GGC CGA TGC TGC ACG CCC CAG CTG ACC AGG ACT GTGAAG ATG CGG TTC 1128 Gly Arg Cys Cys Thr Pro Gln Leu Thr Arg Thr Val LysMet Arg Phe 320 325 330 335 CGC TGC GAA GAT GGG GAG ACA TTT TCC AAG AACGTC ATG ATG ATC CAG 1176 Arg Cys Glu Asp Gly Glu Thr Phe Ser Lys Asn ValMet Met Ile Gln 340 345 350 TCC TGC AAA TGC AAC TAC AAC TGC CCG CAT GCCAAT GAA GCA GCG TTT 1224 Ser Cys Lys Cys Asn Tyr Asn Cys Pro His Ala AsnGlu Ala Ala Phe 355 360 365 CCC TTC TAC AGG CTG TTC AAT GAC ATT CAC AAATTT AGG GAC 1266 Pro Phe Tyr Arg Leu Phe Asn Asp Ile His Lys Phe Arg Asp370 375 380 TAAATGCTAC CTGGGTTTCC AGGGCACACC TAGACAAACA AGGGAGAAGAGTGTCAGA 1326 CAGAATCATG GAGAAAATGG GCGGGGGTGG TGTGGGTGAT GGGACTCATTGTAGAAAG 1386 AGCCTTCTCA TTCTTGAGGA GCATTAAGGT AT 1418 381 amino acidsamino acid linear protein misc_feature “Human Cyr61 amino acid sequence”4 Met Ser Ser Arg Ile Ala Arg Ala Leu Ala Leu Val Val Thr Leu Leu 1 5 1015 His Leu Thr Arg Leu Ala Leu Ser Thr Cys Pro Ala Ala Cys His Cys 20 2530 Pro Leu Glu Ala Pro Lys Cys Ala Pro Gly Val Gly Leu Val Arg Asp 35 4045 Gly Cys Gly Cys Cys Lys Val Cys Ala Lys Gln Leu Asn Glu Asp Cys 50 5560 Ser Lys Thr Gln Pro Cys Asp His Thr Lys Gly Leu Glu Cys Asn Phe 65 7075 80 Gly Ala Ser Ser Thr Ala Leu Lys Gly Ile Cys Arg Ala Gln Ser Glu 8590 95 Gly Arg Pro Cys Glu Tyr Asn Ser Arg Ile Tyr Gln Asn Gly Glu Ser100 105 110 Phe Gln Pro Asn Cys Gln His Gln Cys Thr Cys Ile Asp Gly AlaVal 115 120 125 Gly Cys Ile Pro Leu Cys Pro Gln Glu Leu Ser Leu Pro AsnLeu Gly 130 135 140 Cys Pro Asn Pro Arg Leu Val Lys Val Thr Gly Gln CysCys Glu Glu 145 150 155 160 Trp Val Cys Asp Glu Asp Ser Ile Lys Asp ProMet Glu Asp Gln Asp 165 170 175 Gly Leu Leu Gly Lys Glu Leu Gly Phe AspAla Ser Glu Val Glu Leu 180 185 190 Thr Arg Asn Asn Glu Leu Ile Ala ValGly Lys Gly Arg Ser Leu Lys 195 200 205 Arg Leu Pro Val Phe Gly Met GluPro Arg Ile Leu Tyr Asn Pro Leu 210 215 220 Gln Gly Gln Lys Cys Ile ValGln Thr Thr Ser Trp Ser Gln Cys Ser 225 230 235 240 Lys Thr Cys Gly ThrGly Ile Ser Thr Arg Val Thr Asn Asp Asn Pro 245 250 255 Glu Cys Arg LeuVal Lys Glu Thr Arg Ile Cys Glu Val Arg Pro Cys 260 265 270 Gly Gln ProVal Tyr Ser Ser Leu Lys Lys Gly Lys Lys Cys Ser Lys 275 280 285 Thr LysLys Ser Pro Glu Pro Val Arg Phe Thr Tyr Ala Gly Cys Leu 290 295 300 SerVal Lys Lys Tyr Arg Pro Lys Tyr Cys Gly Ser Cys Val Asp Gly 305 310 315320 Arg Cys Cys Thr Pro Gln Leu Thr Arg Thr Val Lys Met Arg Phe Arg 325330 335 Cys Glu Asp Gly Glu Thr Phe Ser Lys Asn Val Met Met Ile Gln Ser340 345 350 Cys Lys Cys Asn Tyr Asn Cys Pro His Ala Asn Glu Ala Ala PhePro 355 360 365 Phe Tyr Arg Leu Phe Asn Asp Ile His Lys Phe Arg Asp 370375 380 2267 base pairs nucleic acid single linear DNA misc_feature“Fisp12 cDNA coding sequence” 5 GAATTCCGCC GACAACCCCA GACGCCACCGCCTGGAGCGT CCAGACACCA ACCTCCGCCC 60 CTGTCCGAAT CCAGGCTCCA GCCGCGCCTCTCGTCGCCTC TGCACCCTGC TGTGCATCCT 120 CCTACCGCGT CCCGATCATG CTCGCCTCCGTCGCAGGTCC CATCAGCCTC GCCTTGGTGC 180 TCCTCGCCCT CTGCACCCGG CCTGCTACGGGCCAGGACTG CAGCGCGCAA TGTCAGTGCG 240 CAGCCGAAGC AGCGCCGCAC TGCCCCGCCGGCGTGAGCCT GGTGCTGGAC GGCTGCGGCT 300 GCTGCCGCGT CTGCGCCAAG CAGCTGGGAGAACTGTGTAC GGAGCGTGAC CCCTGCGACC 360 CACACAAGGG CCTCTTCTGC GATTTCGGCTCCCCCGCCAA CCGCAAGATT GGAGTGTGCA 420 CTGCCAAAGA TGGTGCACCC TGTGTCTTCGGTGGGTCGGT GTACCGCAGC GGTGAGTCCT 480 TCCAAAGCAG CTGCAAATAC CAATGCACTTGCCTGGATGG GGCCGTGGGC TGCGTGCCCC 540 TATGCAGCAT GGACGTGCGC CTGCCCAGCCCTGACTGCCC CTTCCCGAGA AGGGTCAAGC 600 TGCCTGGGAA ATGCTGCAAG GAGTGGGTGTGTGACGAGCC CAAGGACCGC ACAGCAGTTG 660 GCCCTGCCCT AGCTGCCTAC CGACTGGAAGACACATTTGG CCCAGACCCA ACTATGATGC 720 GAGCCAACTG CCTGGTCCAG ACCACAGAGTGGAGCGCCTG TTCTAAGACC TGTGGAATGG 780 GCATCTCCAC CCGAGTTACC AATGACAATACCTTCTGCAG ACTGGAGAAG CAGAGCCGCC 840 TCTGCATGGT CAGGCCCTGC GAAGCTGACCTGGAGGAAAA CATTAAGAAG GGCAAAAAGT 900 GCATCCGGAC ACCTAAAATC GCCAAGCCTGTCAAGTTTGA GCTTTCTGGC TGCACCAGTG 960 TGAAGACATA CAGGGCTAAG TTCTGCGGGGTGTGCACAGA CGGCCGCTGC TGCACACCGC 1020 ACAGAACCAC CACTCTGCCA GTGGAGTTCAAATGCCCCGA TGGCGAGATC ATGAAAAAGA 1080 ATATGATGTT CATCAAGACC TGTGCCTGCCATTACAACTG TCCTGGGGAC AATGACATCT 1140 TTGAGTCCCT GTACTACAGG AAGATGTACGGAGACATGGC GTAAAGCCAG GAAGTAAGGG 1200 ACACGAACTC ATTAGACTAT AACTTGAACTGAGTTGCATC TCATTTTCTT CTGTAAAAAC 1260 AATTACAGTA GCACATTAAT TTAAATCTGTGTTTTTAACT ACCGTGGGAG GAACTATCCC 1320 ACCAAAGTGA GAACGTTATG TCATGGCCATACAAGTAGTC TGTCAACCTC AGACACTGGT 1380 TTCGAGACAG TTTACACTTG ACAGTTGTTCATTAGCGCAC AGTGCCAGAA CGCACACTGA 1440 GGTGAGTCTC CTGGAACAGT GGAGATGCCAGGAGAAAGAA AGACAGGTAC TAGCTGAGGT 1500 TATTTTAAAA GCAGCAGTGT GCCTACTTTTTGGAGTGTAA CCGGGGAGGG AAATTATAGC 1560 ATGCTTGCAG ACAGACCTGC TCTAGCGAGAGCTGAGCATG TGTCCTCCAC TAGATGAGGC 1620 TGAGTCCAGC TGTTCTTTAA GAACAGCAGTTTCAGCCTCT GACCATTCTG ATTCCAGTGA 1680 CACTTGTCAG GAGTCAGAGC CTTGTCTGTTAGACTGGACA GCTTGTGGCA AGTAAGTTTG 1740 CCTGTAACAA GCCAGATTTT TATTGATATTGTAAATATTG TGGATATATA TATATATATA 1800 TATATTTGTA CAGTTATCTA AGTTAATTTAAAGTCATTTG TTTTTGTTTT AAGTGCTTTT 1860 GGGATTTTAA ACTGATAGCC TCAAACTCCAAACACCATAG GTAGGACACG AAGCTTATCT 1920 GTGATTCAAA ACAAAGGAGA TACTGCAGTGGGAATTGTGA CCTGAGTGAC TCTCTGTCAG 1980 AACAAACAAA TGCTGTGCAG GTGATAAAGCTATGTATTGG AAGTCAGATT TCTAGTAGGA 2040 AATGTGGTCA AATCCCTGTT GGTGAACAAATGGCCTTTAT TAAGAAATGG CTGGCTCAGG 2100 GTAAGGTCCG ATTCCTACCA GGAAGTGCTTGCTGCTTCTT TGATTATGAC TGGTTTGGGG 2160 TGGGGGGCAG TTTATTTGTT GAGAGTGTGACCAAAAGTTA CATGTTTGCA CCTTTCTAGT 2220 TGAAAATAAA GTATATATAT ATTTTTTATATGAAAAAAAA GGAATTC 2267 348 amino acids amino acid single linear proteinmisc_feature “Fisp12 amino acid sequence” 6 Met Leu Ala Ser Val Ala GlyPro Ile Ser Leu Ala Leu Val Leu Leu 1 5 10 15 Ala Leu Cys Thr Arg ProAla Thr Gly Gln Asp Cys Ser Ala Gln Cys 20 25 30 Gln Cys Ala Ala Glu AlaAla Pro His Cys Pro Ala Gly Val Ser Leu 35 40 45 Val Leu Asp Gly Cys GlyCys Cys Arg Val Cys Ala Lys Gln Leu Gly 50 55 60 Glu Leu Cys Thr Glu ArgAsp Pro Cys Asp Pro His Lys Gly Leu Phe 65 70 75 80 Cys Asp Phe Gly SerPro Ala Asn Arg Lys Ile Gly Val Cys Thr Ala 85 90 95 Lys Asp Gly Ala ProCys Val Phe Gly Gly Ser Val Tyr Arg Ser Gly 100 105 110 Glu Ser Phe GlnSer Ser Cys Lys Tyr Gln Cys Thr Cys Leu Asp Gly 115 120 125 Ala Val GlyCys Val Pro Leu Cys Ser Met Asp Val Arg Leu Pro Ser 130 135 140 Pro AspCys Pro Phe Pro Arg Arg Val Lys Leu Pro Gly Lys Cys Cys 145 150 155 160Lys Glu Trp Val Cys Asp Glu Pro Lys Asp Arg Thr Ala Val Gly Pro 165 170175 Ala Leu Ala Ala Tyr Arg Leu Glu Asp Thr Phe Gly Pro Asp Pro Thr 180185 190 Met Met Arg Ala Asn Cys Leu Val Gln Thr Thr Glu Trp Ser Ala Cys195 200 205 Ser Lys Thr Cys Gly Met Gly Ile Ser Thr Arg Val Thr Asn AspAsn 210 215 220 Thr Phe Cys Arg Leu Glu Lys Gln Ser Arg Leu Cys Met ValArg Pro 225 230 235 240 Cys Glu Ala Asp Leu Glu Glu Asn Ile Lys Lys GlyLys Lys Cys Ile 245 250 255 Arg Thr Pro Lys Ile Ala Lys Pro Val Lys PheGlu Leu Ser Gly Cys 260 265 270 Thr Ser Val Lys Thr Tyr Arg Ala Lys PheCys Gly Val Cys Thr Asp 275 280 285 Gly Arg Cys Cys Thr Pro His Arg ThrThr Thr Leu Pro Val Glu Phe 290 295 300 Lys Cys Pro Asp Gly Glu Ile MetLys Lys Asn Met Met Phe Ile Lys 305 310 315 320 Thr Cys Ala Cys His TyrAsn Cys Pro Gly Asp Asn Asp Ile Phe Glu 325 330 335 Ser Leu Tyr Tyr ArgLys Met Tyr Gly Asp Met Ala 340 345 2075 base pairs nucleic acid singlelinear DNA misc_feature “CTGF cDNA coding sequence” 7 CCCGGCCGACAGCCCCGAGA CGACAGCCCG GCGCGTCCCG GTCCCCACCT CCGACCACCG 60 CCAGCGCTCCAGGCCCCGCG CTCCCCGCTC GCCGCCACCG CGCCCTCCGC TCCGCCCGCA 120 GTGCCAACCATGACCGCCGC CAGTATGGGC CCCGTCCGCG TCGCCTTCGT GGTCCTCCTC 180 GCCCTCTGCAGCCGGCCGGC CGTCGGCCAG AACTGCAGCG GGCCGTGCCG GTGCCCGGAC 240 GAGCCGGCGCCGCGCTGCCC GGCGGGCGTG AGCCTCGTGC TGGACGGCTG CGGCTGCTGC 300 CGCGTCTGCGCCAAGCAGCT GGGCGAGCTG TGCACCGAGC GCGACCCCTG CGACCCGCAC 360 AAGGGCCTCTTCTGTGACTT CGGCTCCCCG GCCAACCGCA AGATCGGCGT GTGCACCGCC 420 AAAGATGGTGCTCCCTGCAT CTTCGGTGGT ACGGTGTACC GCAGCGGAGA GTCCTTCCAG 480 AGCAGCTGCAAGTACCAGTG CACGTGCCTG GACGGGGCGG TGGGCTGCAT GCCCCTGTGC 540 AGCATGGACGTTCGTCTGCC CAGCCCTGAC TGCCCCTTCC CGAGGAGGGT CAAGCTGCCC 600 GGGAAATGCTGCGAGGAGTG GGTGTGTGAC GAGCCCAAGG ACCAAACCGT GGTTGGGCCT 660 GCCCTCGCGGCTTACCGACT GGAAGACACG TTTGGCCCAG ACCCAACTAT GATTAGAGCC 720 AACTGCCTGGTCCAGACCAC AGAGTGGAGC GCCTGTTCCA AGACCTGTGG GATGGGCATC 780 TCCACCCGGGTTACCAATGA CAACGCCTCC TGCAGGCTAG AGAAGCAGAG CCGCCTGTGC 840 ATGGTCAGGCCTTGCGAAGC TGACCTGGAA GAGAACATTA AGAAGGGCAA AAAGTGCATC 900 CGTACTCCCAAAATCTCCAA GCCTATCAAG TTTGAGCTTT CTGGCTGCAC CAGCATGAAG 960 ACATACCGAGCTAAATTCTG TGGAGTATGT ACCGACGGCC GATGCTGCAC CCCCCACAGA 1020 ACCACCACCCTGCCGGTGGA GTTCAAGTGC CCTGACGGCG AGGTCATGAA GAAGAACATG 1080 ATGTTCATCAAGACCTGTGC CTGCCATTAC AACTGTCCCG GAGACAATGA CATCTTTGAA 1140 TCGCTGTACTACAGGAAGAT GTACGGAGAC ATGGCATGAA GCCAGAGAGT GAGAGACATT 1200 AACTCATTAGACTGGAACTT GAACTGATTC ACATCTCATT TTTCCGTAAA AATGATTTCA 1260 GTAGCACAAGTTATTTAAAT CTGTTTTTCT AACTGGGGGA AAAGATTCCC ACCCAATTCA 1320 AAACATTGTGCCATGTCAAA CAAATAGTCT ATCTTCCCCA GACACTGGTT TGAAGAATGT 1380 TAAGACTTGACAGTGGAACT ACATTAGTAC ACAGCACCAG AATGTATATT AAGGTGTGGC 1440 TTTAGGAGCAGTGGGAGGGT ACCGGCCCGG TTAGTATCAT CAGATCGACT CTTATACGAG 1500 TAATATGCCTGCTATTTGAA GTGTAATTGA GAAGGAAAAT TTTAGCGTGC TCACTGACCT 1560 GCCTGTAGCCCCAGTGACAG CTAGGATGTG CATTCTCCAG CCATCAAGAG ACTGAGTCAA 1620 GTTGTTCCTTAAGTCAGAAC AGCAGACTCA GCTCTGACAT TCTGATTCGA ATGACACTGT 1680 TCAGGAATCGGAATCCTGTC GATTAGACTG GACAGCTTGT GGCAAGTGAA TTTGCCTGTA 1740 ACAAGCCAGATTTTTTAAAA TTTATATTGT AAATATTGTG TGTGTGTGTG TGTGTGTATA 1800 TATATATATATATGTACAGT TATCTAAGTT AATTTAAAGT TGTTTGTGCC TTTTTATTTT 1860 TGTTTTTAATGCTTTGATAT TTCAATGTTA GCCTCAATTT CTGAACACCA TAGGTAGAAT 1920 GTAAAGCTTGTCTGATCGTT CAAAGCATGA AATGGATACT TATATGGAAA TTCTGCTCAG 1980 ATAGAATGACAGTCCGTCAA AACAGATTGT TTGCAAAGGG GAGGCATCAG TGTCTTGGCA 2040 GGCTGATTTCTAGGTAGGAA ATGTGGTAGC TCACG 2075 349 amino acids amino acid singlelinear protein misc_feature “CTGF amino acid sequence” 8 Met Thr Ala AlaSer Met Gly Pro Val Arg Val Ala Phe Val Val Leu 1 5 10 15 Leu Ala LeuCys Ser Arg Pro Ala Val Gly Gln Asn Cys Ser Gly Pro 20 25 30 Cys Arg CysPro Asp Glu Pro Ala Pro Arg Cys Pro Ala Gly Val Ser 35 40 45 Leu Val LeuAsp Gly Cys Gly Cys Cys Arg Val Cys Ala Lys Gln Leu 50 55 60 Gly Glu LeuCys Thr Glu Arg Asp Pro Cys Asp Pro His Lys Gly Leu 65 70 75 80 Phe CysAsp Phe Gly Ser Pro Ala Asn Arg Lys Ile Gly Val Cys Thr 85 90 95 Ala LysAsp Gly Ala Pro Cys Ile Phe Gly Gly Thr Val Tyr Arg Ser 100 105 110 GlyGlu Ser Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys Leu Asp 115 120 125Gly Ala Val Gly Cys Met Pro Leu Cys Ser Met Asp Val Arg Leu Pro 130 135140 Ser Pro Asp Cys Pro Phe Pro Arg Arg Val Lys Leu Pro Gly Lys Cys 145150 155 160 Cys Glu Glu Trp Val Cys Asp Glu Pro Lys Asp Gln Thr Val ValGly 165 170 175 Pro Ala Leu Ala Ala Tyr Arg Leu Glu Asp Thr Phe Gly ProAsp Pro 180 185 190 Thr Met Ile Arg Ala Asn Cys Leu Val Gln Thr Thr GluTrp Ser Ala 195 200 205 Cys Ser Lys Thr Cys Gly Met Gly Ile Ser Thr ArgVal Thr Asn Asp 210 215 220 Asn Ala Ser Cys Arg Leu Glu Lys Gln Ser ArgLeu Cys Met Val Arg 225 230 235 240 Pro Cys Glu Ala Asp Leu Glu Glu AsnIle Lys Lys Gly Lys Lys Cys 245 250 255 Ile Arg Thr Pro Lys Ile Ser LysPro Ile Lys Phe Glu Leu Ser Gly 260 265 270 Cys Thr Ser Met Lys Thr TyrArg Ala Lys Phe Cys Gly Val Cys Thr 275 280 285 Asp Gly Arg Cys Cys ThrPro His Arg Thr Thr Thr Leu Pro Val Glu 290 295 300 Phe Lys Cys Pro AspGly Glu Val Met Lys Lys Asn Met Met Phe Ile 305 310 315 320 Lys Thr CysAla Cys His Tyr Asn Cys Pro Gly Asp Asn Asp Ile Phe 325 330 335 Glu SerLeu Tyr Tyr Arg Lys Met Tyr Gly Asp Met Ala 340 345 25 base pairsnucleic acid single linear cDNA 9 GGGGATCTGT GACGAGCCCA AGGAC 25 26 basepairs nucleic acid single linear cDNA 10 GGGAATTCGA CCAGGCAGTT GGCTCG 2626 base pairs nucleic acid single linear cDNA 11 GGGGATCCTG TGATGAAGACAGCATT 26 26 base pairs nucleic acid single linear cDNA 12 GGGAATTCAACGATGCATTT CTGGCC 26 21 amino acids amino acid not relevant not relevantpeptide 13 Asp Gly Cys Gly Cys Cys Lys Val Cys Ala Lys Gln Leu Asn GluAsp 1 5 10 15 Cys Ser Lys Thr Gln 20 21 amino acids amino acid notrelevant not relevant peptide 14 Pro Asn Cys Lys His Gln Cys Thr Cys IleAsp Gly Ala Val Gly Cys 1 5 10 15 Ile Pro Leu Cys Pro 20 24 amino acidsamino acid not relevant not relevant peptide 15 Cys Ile Val Gln Thr ThrSer Trp Ser Gln Cys Ser Lys Ser Cys Gly 1 5 10 15 Thr Gly Ile Ser ThrArg Val Thr 20 26 amino acids amino acid not relevant not relevantpeptide 16 Ile Ser Thr Arg Val Thr Asn Asp Asn Pro Glu Cys Arg Leu ValLys 1 5 10 15 Glu Thr Arg Ile Cys Glu Val Arg Pro Cys 20 25 21 aminoacids amino acid not relevant not relevant peptide 17 Lys Tyr Cys GlySer Cys Val Asp Gly Arg Cys Cys Thr Pro Leu Gln 1 5 10 15 Thr Arg ThrVal Lys 20

What is claimed is:
 1. Isolated and purified Cyr61, and biologicallyactive fragments, variants, analogs, homologs, and derivatives thereof.2. An isolated and purified polypeptide having an amino acid sequence asset forth in SEQ ID NO: 4, and fragments, variants, homologs, analogs,and derivatives thereof.
 3. The polypeptide according to claim 2 whereinsaid polypeptide is immunogenic.
 4. A polypeptide according to claim 2wherein said polypeptide is covalently modified.
 5. A polypeptideaccording to claim 4 wherein said covalent modification comprises thecovalent attachment of polyethylene glycol.
 6. The polypeptide of claim4 wherein said modification is a fusion with all or part of a differentpolypeptide.
 7. An antibody that specifically binds to a polypeptideaccording to claim
 1. 8. An antibody according to claim 7 wherein saidantibody is a monoclonal antibody.
 9. A pharmaceutical compositioncomprising a biologically effective amount of the polypeptide accordingto claim 1 and a pharmaceutically acceptable adjuvant, diluent. orcarrier.
 10. A purified and isolated polynucleotide encoding Cyr61. 11.A polynucleotide according to claim 10 wherein said Cyr61 is humanCyr61, and fragments, variants, homologs, analogs, and derivativesthereof.
 12. A purified and isolated polynucleotide encoding apolypeptide having an amino acid sequence as set forth in SEQ ID NO: 4,and fragments, variants, homologs, analogs, and derivatives thereof. 13.A purified and isolated polynucleotide according to claim 12 whereinsaid polypeptide encodes a subsequence of the amino acid sequence setforth in SEQ ID NO:
 4. 14. A purified and isolated polynucleotide havingthe sequence set forth in SEQ ID NO:
 3. 15. A purified and isolatedpolynucleotide that hybridizes under stringent conditions to apolynucleotide according to claim
 10. 16. A vector comprising apolynucleotide according to claim
 10. 17. A host cell transformed ortransfected with a polynucleotide according to claim
 10. 18. A method ofmaking a Cyr61 polypeptide, comprising the steps of: (a) Culturing ahost cell according to claim 15 under suitable nutrient conditions, and(b) purifying said polypeptide from said host cell or from a growthmedium of said host cell.
 19. A method of purifying human Cyr61comprising the steps of: (a) obtaining a biomaterial containing humanCyr61, (b) exposing said biomaterial to a Cyr61-specific biomoleculeselected from the group consisting of anti-Cyr61 antibodies and α_(v)β₃integrin; (c) specifically binding said human Cyr61 to saidCyr61-specific biomolecule: and (c) eluting said human Cyr61, therebypurifying said human Cyr61.
 20. The method according to claim 19 whereinsaid source comprises human cells.
 21. The method according to claim 19wherein said human Cyr61-specific biomolecule is an anti-human Cyr61antibody.
 22. A method of screening for a modulator of angiogenesiscomprising the steps of: (a) contacting a first biological samplecapable of undergoing angiogenesis with a biologically effective amountof an ECM signalling molecule-related biomaterial and a suspectedmodulator; (b) separately contacting a second biological sample with abiologically effective amount of an ECM signalling molecule-relatedbiomaterial, thereby providing a control; (c) measuring the level ofangiogenesis resulting from step (a) and from step (b); and (d)comparing the levels of angiogenesis measured in step (c), whereby amodulator of angiogenesis is identified by its ability to alter thelevel of angiogenesis when compared to the control of step (b).
 23. Themethod according to claim 22 wherein said modulator is an inhibitor ofangiogenesis and further wherein said inhibitor is identified by itsability to decrease said level of angiogenesis when compared to thecontrol of step (b).
 24. The method according to claim 22 wherein saidECM signalling molecule is Cyr61, and fragments, variants, homologs,analogs, and derivatives thereof.
 25. A method of screening for amodulator of angiogenesis comprising the steps of: (a) preparing a firstimplant comprising Cyr61 and a second implant comprising Cyr61 and asuspected modulator of Cyr61; (b) implanting said first implant in afirst cornea of a test animal and said second implant in a second corneaof said test animal: (c) measuring the development of blood vessels insaid first and second corneas; and (d) comparing the levels of bloodvessel development measured in step (c) whereby a modulator ofangiogenesis is identified by its ability to alter the level of bloodvessel development in said first cornea when compared to the bloodvessel development in said second cornea.
 26. A method of screening fora modulator of chondrogenesis comprising the steps of: (a) contacting afirst biological sample capable of undergoing chondrogenesis with abiologically effective amount of an ECM signalling molecule-relatedbiomaterial and a suspected modulator, (b) separately contacting asecond biological sample capable of undergoing chondrogenesis with abiologically effective amount of an ECM signalling molecule-relatedbiomaterial, thereby providing a control: (c) measuring the level ofchondrogenesis resulting from step (a) and from step (b): and (d)comparing the levels of chondrogenesis measured in step (c), whereby amodulator of chondrogenesis is identified by its ability to alter thelevel of chondrogenesis when compared to the control of step (b). 27.The method according to claim 26 wherein said modulator is an inhibitorof chondrogenesis and further wherein said inhibitor is identified byits ability to decrease said level of chondrogenesis when compared tothe control of step (b).
 28. The method according to claim 26 whereinsaid ECM signalling molecule-related biomaterial is selected from thegroup consisting of a human Cyr61, a human Cyr61 fragment, a human Cyr61analog, a human Cyr61 derivative, an antibody specifically recognizinghuman Cyr61, an inhibitor peptide. mannose-6-phosphate, heparin, andtenascin.
 29. A method of screening for a modulator of oncogenesiscomprising the steps of: (a) inducing a first tumor and a second tumor:(b) administering a biologically effective amount of an ECM signallingmolecule-related biomaterial and a suspected modulator to said firsttumor; (c) separately administering a biologically effective amount ofan ECM signalling molecule-related biomaterial to said second tumor,thereby providing a control; (d) measuring the level of oncogenesisresulting from step (b) and from step (c); and (e) comparing the levelsof oncogenesis measured in step (d), whereby a modulator of oncogenesisis identified by its ability to alter the level of oncogenesis whencompared to the control of step (c).
 30. The method according to claim29 wherein said modulator is an inhibitor of oncogenesis and furtherwherein said inhibitor is identified by its ability to decrease saidlevel of oncogenesis when compared to the control of step (b).
 31. Amethod for treating a solid tumor comprising the step of delivering atherapeutically effective amount of a Cyr61 inhibitor to an individual,thereby inhibiting the neovascularization of said tumor.
 32. The methodaccording to claim 31 wherein said inhibitor is selected from the groupconsisting of inhibitor peptides and cytotoxins.
 33. The methodaccording to claim 31 wherein said inhibitor is a cytotoxin attached toCyr61.
 34. A method of screening for a modulator of cell adhesioncomprising the steps of: (a) preparing a surface compatible with celladherence; (b) separately placing first and second biological samples,each sample capable of undergoing cell adhesion, on said surface; (c)contacting a first biological sample with a suspected modulator and abiologically effective amount of an ECM signalling molecule-relatedbiomaterial selected from the group consisting of a human Cyr61, a humanCyr61 fragment, a human Cyr61 analog, and a human Cyr61 derivative; (d)separately contacting a second biological sample with a biologicallyeffective amount of an ECM signalling molecule-related biomaterialselected from the group consisting of a human Cyr61, a human Cyr6fragment, a human Cyr61 analog, and a human Cyr61 derivative. therebyproviding a control; (e) measuring the level of cell adhesion resultingfrom step (c) and from step (d); and (f) comparing the levels of celladhesion measured in step (e). whereby a modulator of cell adhesion isidentified by its ability to alter the level of cell adhesion whencompared to the control of step (d).
 35. A method of screening for amodulator of cell migration comprising the steps of: (a) forming a gelmatrix comprising Cyr61 and a suspected modulator of cell migration; (b)preparing a control gel matrix comprising Cyr61; (c) seeding endothelialcells capable of undergoing cell migration onto the gel matrix of step(a) and the control gel matrix of step (b); (d) incubating saidendothelial cells; (e) measuring the levels of cell migration byinspecting the interior of said gel matrix and said control gel matrixfor cells; (f) comparing the levels of cell migration measured in step(e). whereby a modulator of cell migration is identified by its abilityto alter the level of cell migration in the gel matrix when compared tothe level of cell migration in the control gel matrix.
 36. The methodaccording to claim 35 wherein said endothelial cells are human cells.37. The method according to claim 35 wherein said matrix is selectedfrom the group consisting of Matrigel, collagen, and fibrin.
 38. Themethod according to claim 35 wherein said inspecting step comprisesmicroscopic examination.
 39. An in vitro method of screening for cellmigration comprising the steps of: (a) forming a first gelatinizedfilter and a second gelatinized filter, each filter having two sides;(b) contacting a first side of each said filter with endothelial cellscapable of undergoing cell migration, thereby adhering said cells toeach said filter; (c) applying an ECM signalling molecule and asuspected modulator of cell migration to a second side of said firstgelatinized filter and an ECM signalling molecule to a second side ofsaid second gelatinized filter; (d) incubating each said filter; (e)detecting cells on said second side of each said filter; and (f)comparing the presence of cells on said second side of said firstgelatinized filter with the presence of cells on said second side ofsaid second gelatinized filter, whereby a modulator of cell migration isidentified by its ability to alter the level of cell migration measuredon said first gelatinized filter when compared to the cell migrationmeasured on said second gelatinized filter.
 40. The method according toclaim 39 wherein said endothelial cells are human microvascularendothelial cells.
 41. The method according to claim 39 wherein said ECMsignalling molecule is human Cyr61.
 42. The method according to claim 39further comprising the step of placing each said filter in a Boydenchamber.
 43. An in vivo method of screening for a modulator of cellmigration comprising the steps of: (a) removing a first central portionof a first biocompatible sponge and a second central portion of a secondbiocompatible sponge: (b) applying an ECM signalling molecule and asuspected modulator to said first central portion and an ECM signallingmolecule to said second central portion; (c) reassociating said firstcentral portion with said first biocompatible sponge and said secondcentral portion with said second biocompatible sponge; (d) attaching afirst filter to a first side of said first biocompatible sponge and asecond filter to a second side of said first biocompatible sponge; (e)attaching a third filter to a first side of said second biocompatiblesponge and a fourth filter to a second side of said second biocompatiblesponge; (f) implanting each of said biocompatible sponges, eachbiocompatible sponge comprising said central portion and said filters,in a test animal; (e) removing each said sponge following a period ofincubation; (f) measuring the cells found within each of saidbiocompatible sponges; and (g) comparing the presence of cells in saidfirst biocompatible sponge with the presence of cells in said secondbiocompatible sponge, whereby a modulator of cell migration isidentified by its ability to alter the level of cell migration measuredusing said first biocompatible sponge when compared to the cellmigration measured using said second biocompatible sponge.
 44. Themethod according to claim 43 wherein said ECM signalling molecule ishuman Cyr61.
 45. The method according to claim 43 wherein said ECMsignalling molecule is associated with Hydron.
 46. The method accordingto claim 43 further comprising the step of providing a radiolabel tosaid test animal prior to removing said first and second biocompatiblesponges and wherein said detecting step comprises the detection of saidradiolabel in said first and second biocompatible sponges.
 47. A methodfor modulating hemostasis comprising the step of administering an ECMsignalling molecule in a pharmaceutically acceptable adjuvant, diluentor carrier.
 48. The method according to claim 47 wherein said ECMsignalling molecule is human Cyr61.
 49. A method of inducing woundhealing in a tissue comprising contacting wounded tissue with anangiogenically effective amount of Cyr61.
 50. A method of inducing woundhealing in a tissue comprising the steps of: (a) introducing a nucleicacid comprising a control expression sequence operably linked to an ECMsignalling molecule into the cells of a wounded tissue: and (b)controlling the expression of said coding region, thereby inducing woundhealing.
 51. The method according to claim 50 wherein said ECMsignalling molecule is human Cyr61.
 52. The method according to claim 50wherein said nucleic acid comprises a vector selected from the groupconsisting of a Herpesvirus, an Adenovirus, an Adeno-associated Virus, aCytomegalovirus, a Baculovirus, a retrovirus, and a Vaccinia Virus, andwherein said vector comprises an ECM signalling molecule coding region.53. The method according to claim 50 wherein said wounded tissue isselected from the group consisting of skin tissue and lung epithelialtissue.
 54. A method of promoting organ regeneration comprising the stepof administering a biologically effective quantity of Cyr61 to ananimal.
 55. A method of improving tissue grafting comprising the step ofadministering to an animal a quantity of Cyr61 effective in improvingthe rate of neovascularization of a tissue graft.
 56. A method forpromoting bone implantation comprising the step of applying abiologically effective amount of an ECM signalling molecule to a boneimplant, thereby promoting bone implantation.
 57. A method for promotingprosthesis implantation comprising the steps of: (a) applying abiologically effective amount of an ECM signalling molecule to abiocompatible wrap such as a biodegradable gauze: and (b) contacting thewrap with a prosthesis; and (c) implanting said prosthesis, therebypromoting prosthesis implantation.
 58. A method of screening for amodulator of cell proliferation comprising the steps of: (a) contactinga first biological sample capable of undergoing cell proliferation witha suspected modulator and a biologically effective amount of an ECMsignalling molecule-related biomaterial selected from the groupconsisting of a human Cyr61, a human Cyr61 fragment, a human Cyr61analog, and a human Cyr61 derivative: (b) separately contacting a secondbiological sample with a biologically effective amount of an ECMsignalling molecule-related biomaterial selected from the groupconsisting of a human Cyr61, a human Cyr61 fragment, a human Cyr61analog, and a human Cyr61 derivative, thereby providing a control; (c)incubating said first and second biological samples; (d) measuring thelevel of cell proliferation resulting from step (c); and (e) comparingthe levels of cell proliferation measured in step (d), whereby amodulator of cell proliferation is identified by its ability to alterthe level of cell adhesion when compared to the control of step (b). 59.A method for expanding a population of undifferentiated hematopoieticstem cells in culture, comprising the steps of: (a) obtaininghematopoietic stem cells from a donor; and (b) culturing said cellsunder suitable nutrient conditions in the presence of a biologicallyeffective amount of Cyr61.
 60. A method of screening for a mitogencomprising the steps of: (a) plating cells capable of undergoing cellproliferation; (b) contacting a first portion of said cells with asolution comprising Cyr61 and a suspected mitogen; (c) contacting asecond portion of said cells with a solution comprising Cyr61, therebyproviding a control: (c) incubating said cells; (d) detecting the growthof said first portion of cells and said second portion of said cells;and (e) comparing growth of said first and second portions of cells.whereby a mitogen is identified by its ability to induce greater growthin said first portion of cells when compared to the growth of saidsecond portion of cells.
 61. The method according to claim 60 whereinsaid cells are selected from the group consisting of endothelial cellsand fibroblast cells.
 62. The method according to claim 60 furthercomprising contacting said first and second. portions of said cells witha nucleic acid label and detecting the presence of said nucleic acidlabel in said cells.
 63. The method according to claim 62 wherein saidnucleic acid label is [³H]-thymidine.
 64. A kit comprising a polypeptideaccording to claim 2.